Patent application title: Anti-REG4 Antibodies

Abstract:

Antibodies to human REG4 are provided, as well as uses thereof, e.g., in
treatment of proliferative disorders. Also provided is a method of
screening for an antibody that inhibits REG4 bioactivity.

Claims:

1. A binding compound that binds to human REG4 comprising: a) an antibody
light chain variable domain, or antigen binding fragment thereof, having
one or more CDR sequence selected from the group consisting of SEQ ID
NOs: 41-64; and b) an antibody heavy chain variable domain, or antigen
binding fragment thereof, having one or more CDR sequence selected from
the group consisting of SEQ ID NOs: 17-40.

2. The binding compound of claim 1 comprising: a) an antibody light chain
variable domain, or antigen binding fragment thereof, having two or more
CDR sequences selected from the group consisting of SEQ ID NOs: 41-64;
and b) an antibody heavy chain variable domain, or antigen binding
fragment thereof, having two or more CDR sequences selected from the
group consisting of SEQ ID NOs: 17-40.

3. The binding compound of claim 2 comprising: a) an antibody light chain
variable domain, or antigen binding fragment thereof, having three CDR
sequences selected from the group consisting of SEQ ID NOs: 41-64 and b)
an antibody heavy chain variable domain, or antigen binding fragment
thereof, having three CDR sequences selected from the group consisting of
SEQ ID NOs: 17-40.

4. A binding compound that binds to human REG4 comprising: a) a light
chain variable domain, or antigen binding fragment thereof, having at
least one CDRL1 from the group consisting of SEQ ID NOs: 41-48; at least
one CDRL2 from the group consisting of SEQ ID NOs: 49-56; and at least
one CDRL3 from the group consisting of SEQ ID NOs: 57-64; and b) a heavy
chain variable domain, or antigen binding fragment thereof, having at
least one CDRH1 from the group consisting of SEQ ID NOs: 17-24; at least
one CDRH2 from the group consisting of SEQ ID NOs: 25-32; and at least
one CDRH3 from the group consisting of SEQ ID NOs: 33-40.

6. An antibody that is able to block binding of the binding compound of
claim 5 to human REG4 in a cross-blocking assay.

7. An isolated nucleic acid encoding at least one of the light chain
variable domain or heavy chain variable domain of the binding compound of
claim 5.

8. An expression vector comprising the nucleic acid of claim 7 operably
linked to control sequences that are recognized by a host cell when the
host cell is transfected with the vector.

9. A host cell comprising the expression vector of claim 8.

10. A method of producing a polypeptide comprising: culturing the host
cell of claim 9 in culture medium under conditions wherein the nucleic
acid sequence is expressed, thereby producing polypeptides comprising the
light and heavy chain variable domains; and recovering the polypeptides
from the host cell or culture medium.

14. The binding compound of claim 5, wherein the binding compound is an
antibody fragment selected from the group consisting of Fab, Fab',
Fab'-SH, Fv, scFv, F(ab')2, and a diabody.

15. A method of treating a proliferative disorder in a human subject
comprising administering to a subject in need thereof an antibody
specific for REG4, or a antigen binding fragment thereof, in an amount
effective to inhibit at least one biological activity of REG4, wherein
the antibody is the antibody of claim 5.

16. The method of claim 15, wherein the proliferative disorder is a
cancer or a tumor.

17. A method of inhibiting angiogenesis in a human subject comprising
administering to a subject in need thereof an antibody specific for REG4,
or a antigen binding fragment thereof, in an amount effective to inhibit
the biological activity REG4, wherein the antibody is the antibody of
claim 5.

18. A pharmaceutical composition comprising the antibody of claim 5 and a
pharmaceutically acceptable carrier.

19. A method of screening for an inhibitor of REG4 comprising: a)
contacting a cell with a REG4 antagonist; b) measuring at least one
phosphorylation event in the cell; and c) selecting the REG4 inhibitor
that decreases phosphorylation of at least one molecule expressed by the
cell.

20. The method of claim 19, wherein the REG4 antagonist is a REG4
antibody or fragment thereof.

Description:

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This filing is a U.S. patent application which claims benefit of
U.S. Provisional Patent Application No. 61/307,769, filed Feb. 24, 2010,
and U.S. Provisional Patent Application No. 61/261,240, filed Nov. 13,
2009, each of which is hereby incorporated by reference in its entirety
herein.

[0004] The human Regenerating islet-derived Gene ("REG") family of ligands
consists of four secreted and structurally unique proteins that share
sequence similarity with the carbohydrate-binding domain of C-type
lectins. The initial cDNA in this gene family was named REG for its role
in islet of Langerhans regeneration following partial pancreatectomy (now
known as REG Iα). Additional members of the human REG gene family
are regenerating gene homologue (REG Iβ) and pancreatitis-associated
protein (Reg III). All are constitutively expressed in the normal
proximal gastrointestinal tract. While the function of this gene family
is poorly understood, recent data has suggested that REG family members
may function as tissue mitogens. REG Iα is mitogenic for gastric
mucosal cells (Fukui et al. (1998) Gastroenterol. 115:1483-1493), and
pancreatic ductal and beta cells (Zenilman et al. (1996) Gastroenterology
110:1208-1214; Zenilman et al. (1998) Pancreas 17:256-261; Watanabe
(1994) Proc. Nat'l. Acad. Sci. 91:3589-1392). The serum concentration of
REG Iα is significantly increased in many gastrointestinal
malignancies, including gastric and pancreatic adenocarcinoma (Satomura
et al. (1995) J Gastroenterol. 30:643-650). For patients with early stage
colonic adenocarcinoma undergoing surgical resection, REG Iα mRNA
expression alone or co-expression of REG Iα and REG III mRNA by the
carcinoma had an adverse effect on disease free survival that was
independent of tumor stage or site (Macadam et al. (2000) British J
Cancer 83:188-95).

[0005] A novel member of this gene family, REG IV or REG4, which has
significant constitutive expression in the distal gastrointestinal tract
was identified by Hartupee et al. (2001) Biochim. Biophys. Acta
1518:287-293. Through molecular modeling, it was shown that the REG4
protein showed maintenance of the conserved contact surface residues that
cluster on a single face of the 3-dimensional molecule present in all
other members of the REG gene family. This suggests that REG proteins may
share similar physiologic actions. Reg IV is of considerable interest
because of its possible role, along with other members of the Reg gene
family in the pathogenesis of colorectal adenocarcinoma. REG IV is
overexpressed by a majority of colorectal adenocarcimonas. By
differential display, REG IV was among several genes with increased mRNA
expression in several colon cancer cell lines selected for increased in
vitro resistance to a cancer chemotherapeutic agent, 5-FU (Violette et
al. (2003) Int J. Cancer 103:185-193).

[0006] Key to the initiation and progression of cancer is the sustained
activation of tyrosine kinase signaling pathways, which relay signals for
growth, survival, migration and differentiation. Receptor tyrosine
kinases (RTKs) constitute a family of receptors that include epidermal
growth factor receptor (EGFR), insulin receptor (IR), insulin-like growth
factor receptor (IGF1R) and exist as monomers in an inactive state.
Either through mutation or upon extracellular ligand binding, they form
homo or heterodimers, which leads to autophosphorylation of the
intracellular domain and ultimately results in activation of downstream
signaling pathways, including Ras/mitogen activated protein kinase (MAPK)
and phosphatidylinositol-3-kinase (PI3-K) pathways. Given the critical
cell functions regulated by RTKs, both small molecule and monoclonal
antibodies directed against this receptor family have been generated and
are currently used to treat cancer patients {ref}. However, like many
monotherapies, targeting a single RTK pathway is often not sufficient for
complete tumor regression. Consequently, a high percentage of cancers
acquire resistance to these treatments, which necessitates identifying
other key players in cancer biology.

[0008] Many current drugs on the market or in clinical trials are designed
to target one RTK, however, human cancer cells simultaneously activate
numerous growth factor receptors. In addition, since acquired resistance
to EGFR inhibitors can involve upregulation of IGF1R and alternative
growth factor pathways, hitting multiple RTKS simultaneously may delay
drug resistance. Due to the importance of IR in glucose homeostasis, an
ideal anti-IGF strategy would target both IGFIR and IR only in tumor
cells. Given the restricted expression profile of Reg IV in normal
tissues, antagonizing Reg4 activity might represent a way to target IR
only in tumors.

[0009] To date, information about REG4 activity has been limited to
expression in various tumors and cancers. Little has been described about
the biology of REG4 and its modulation of downstream molecules in cancer.
The data below suggests that REG4 plays a significant functional role in
cancer, supported by evidence of modulation of downstream cancer relevant
pathways. Thus, the need exists for improved methods and compositions for
the treatment of proliferative disorders by modulating REG4 activity.
Preferably, such compositions would have a high affinity for the target
molecule, and would be able to modulate REG4 activity at relatively low
doses. Preferably, such methods and compositions would be highly specific
for REG4, and not interfere with the activity of other receptors.
Preferably, such methods and compositions would employ antagonists
suitable for modification for the delivery of cytotoxic payloads to
target cells, but also suitable for non-cytotoxic uses. Preferably, such
methods and compositions would employ antibodies modified to limit their
antigenicity when administered to a subject in need thereof.

SUMMARY OF THE INVENTION

[0010] The present invention meets these needs in the art and more by
providing antagonists of REG4, e.g. REG4 antibodies.

[0011] In one aspect the invention provides binding compounds, such as an
antibody or fragment thereof, including humanized or chimeric recombinant
antibodies, that binds human REG4, comprising an antibody light chain
variable domain, or antigen binding fragment thereof, having at least one
or more CDRs selected from the group consisting of SEQ ID NOs: 41-64 and
a heavy chain variable domain, having at least one or more CDRs selected
from the group consisting of SEQ ID NOs: 17-40.

[0012] In other embodiments the binding compound of the present invention
comprises a light chain variable domain and a heavy chain variable
domain, or the antigen binding fragments thereof, described in the
preceding paragraph.

[0013] In some embodiments, the binding compound comprises a framework
region, wherein the amino acid sequence of the framework region is all or
substantially all of a human immunoglobulin amino acid sequence.

[0014] In some embodiments the light chain variable domain comprises a
sequence selected from the group consisting of SEQ ID NOs: 9-16 or a
variant thereof. In some embodiments the heavy chain variable domain
comprises a sequence selected from the group consisting of SEQ ID NOs:
1-8. In yet a further embodiment, the binding compound comprises a light
chain variable domain and a heavy chain variable domain, or the antigen
binding fragments thereof, described in this paragraph.

[0015] In one embodiment, the invention relates to antibodies or fragments
thereof that are able to block the binding of a binding compound of the
present invention to human REG4 in a cross-blocking assay. In various
embodiments the antibody is able to block binding of human REG4 to an
antibody comprising the CDR sequences of antibodies 1C11, 3C2.3D10,
4C5.3B10, 9F3.3A4, 12E1.3C11, 13E1.1B11, 40F6.3F6, 70A9.3C2, and
86C1.2B7, as disclosed herein.

[0016] In some embodiments, the binding compound of the present invention
comprises a humanized antibody comprising the CDRs, or variants thereof,
selected from the CDRs of the antibodies disclosed herein, in combination
with human germline light chain and heavy chain variable domain framework
sequences in place of the rodent frameworks of the parental antibodies.

[0017] In some embodiments, the binding compound of the present invention
further comprises a heavy chain constant region, wherein the heavy chain
constant region comprises a γ1, γ2, γ3, or γ4
human heavy chain constant region or a variant thereof. In various
embodiments the light chain constant region comprises a lambda or a kappa
human light chain constant region.

[0018] In various embodiments the binding compounds of the present
invention are polyclonal, monoclonal, chimeric, humanized or fully human
antibodies or fragments thereof. The present invention also contemplates
that the antigen binding fragment is an antibody fragment selected from
the group consisting of Fab, Fab', Fab'-SH, Fv, scFv, F(ab')2, and a
diabody.

[0019] In some embodiments, the antibody specific for REG4 is the
humanized or chimeric antibody. The present invention encompasses an
isolated nucleic acid encoding the polypeptide sequence of an antibody
embodiment of the binding compound of the present invention. The nucleic
acid can be in an expression vector operably linked to control sequences
recognized by a host cell transfected with the vector. Also encompassed
is a host cell comprising the vector, and a method of producing a
polypeptide comprising culturing the host cell under conditions wherein
the nucleic acid sequence is expressed, thereby producing the
polypeptide, and recovering the polypeptide from the host cell or medium.

[0020] The present invention encompasses a method of inhibiting a
proliferative response in a human subject comprising administering to a
subject in need thereof an antibody (or a antigen binding fragment
thereof) specific for REG4 in an amount effective to inhibit REG4
activity. In a further embodiment, the antibody can inhibit angiogenesis
associated with a cancer or tumor cell in a human subject.

[0021] The present invention encompasses a pharmaceutical composition
comprising an REG4 antibody or fragment thereof and a pharmaceutically
acceptable carrier.

[0022] The present invention provides a method of screening for an
inhibitor of REG4 comprising: a) contacting a cell with a REG4
antagonist; b) measuring at least one phosphorylation event in the cell;
and c) selecting the REG4 inhibitor that decreases at least one
phosphorylation event in the cell. In certain embodiments, the REG4
antagonist is a REG4 antibody or fragment thereof.

DETAILED DESCRIPTION

[0023] As used herein, including the appended claims, the singular forms
of words such as "a," "an," and "the," include their corresponding plural
references unless the context clearly dictates otherwise. Table 10 below
provides a listing of sequence identifiers used in this application. All
references cited herein are incorporated by reference to the same extent
as if each individual publication, database entry (e.g. Genbank sequences
or GeneID entries), patent application, or patent, was specifically and
individually indicated to be incorporated by reference. Citation of the
references herein is not intended as an admission that any of the
foregoing is pertinent prior art, nor does it constitute any admission as
to the contents or date of these publications or documents.

DEFINITIONS

[0024] The terms "REG4", "Regenerating islet-derived Gene Type IV", and
"REG IV" are well known in the art. The human REG4 nucleotide and
polypeptide sequences are disclosed in e.g., U.S. Pat. No. 5,861,494,
U.S. Pat. No. 6,080,722, and U.S. Pat. No. 7,132,509. GenBank®
deposits of the human REG4 amino (NP--114433) and nucleic acid
(NM--032044) sequences are also available.

[0025] "Proliferative activity" encompasses an activity that promotes,
that is necessary for, or that is specifically associated with, e.g.,
normal cell division, as well as cancer, tumors, dysplasia, cell
transformation, metastasis, and angiogenesis.

[0026] "Administration" and "treatment," as it applies to an animal,
human, experimental subject, cell, tissue, organ, or biological fluid,
refers to contact of an exogenous pharmaceutical, therapeutic, diagnostic
agent, or composition to the animal, human, subject, cell, tissue, organ,
or biological fluid. "Administration" and "treatment" can refer, e.g., to
therapeutic, pharmacokinetic, diagnostic, research, and experimental
methods. Treatment of a cell encompasses contact of a reagent to the
cell, as well as contact of a reagent to a fluid, where the fluid is in
contact with the cell. "Administration" and "treatment" also means in
vitro and ex vivo treatments, e.g., of a cell, by a reagent, diagnostic,
binding composition, or by another cell. "Treatment," as it applies to a
human, veterinary, or research subject, refers to therapeutic treatment,
prophylactic or preventative measures, to research and diagnostic
applications. "Treatment" as it applies to a human, veterinary, or
research subject, or cell, tissue, or organ, encompasses contact of an
agent with animal subject, a cell, tissue, physiological compartment, or
physiological fluid. "Treatment of a cell" also encompasses situations
where the agent contacts REG4, e.g., in the fluid phase or colloidal
phase, but also situations where the agonist or antagonist does not
contact the cell or the receptor.

[0027] As used herein, the term "antibody" refers to any form of antibody
that exhibits the desired biological activity. Thus, it is used in the
broadest sense and specifically covers monoclonal antibodies (including
full length monoclonal antibodies), polyclonal antibodies, multispecific
antibodies (e.g., bispecific antibodies), chimeric antibodies, humanized
antibodies, fully human antibodies, etc. so long as they exhibit the
desired biological activity.

[0028] As used herein, the terms "REG4 binding fragment," "binding
fragment thereof" or "antigen binding fragment thereof" encompass a
fragment or a derivative of an antibody that still substantially retains
its biological activity of inducing REG4 signaling referred to herein as
"REG4 inducing activity." The term "antibody fragment" or REG4 binding
fragment refers to a portion of a full length antibody, generally the
antigen binding or variable region thereof. Examples of antibody
fragments include Fab, Fab', F(ab')2, and Fv fragments; diabodies;
linear antibodies; single-chain antibody molecules, e.g., sc-Fv; and
multispecific antibodies formed from antibody fragments. Typically, a
binding fragment or derivative retains at least 10% of its REG4
antagonist activity. Preferably, a binding fragment or derivative retains
at least 25%, 50%, 60%, 70%, 80%, 90%, 95%, 99% or 100% (or more) of its
REG4 antagonist activity, although any binding fragment with sufficient
affinity to exert the desired biological effect will be useful. It is
also intended that a REG4 binding fragment can include variants having
conservative amino acid substitutions that do not substantially alter its
biologic activity.

[0029] The term "monoclonal antibody", as used herein, refers to an
antibody obtained from a population of substantially homogeneous
antibodies, i.e., the individual antibodies comprising the population are
identical except for possible naturally occurring mutations that may be
present in minor amounts. Monoclonal antibodies are highly specific,
being directed against a single antigenic epitope. In contrast,
conventional (polyclonal) antibody preparations typically include a
multitude of antibodies directed against (or specific for) different
epitopes. The modifier "monoclonal" indicates the character of the
antibody as being obtained from a substantially homogeneous population of
antibodies, and is not to be construed as requiring production of the
antibody by any particular method. For example, the monoclonal antibodies
to be used in accordance with the present invention may be made by the
hybridoma method first described by Kohler et al. (1975) Nature 256: 495,
or may be made by recombinant DNA methods (see, e.g., U.S. Pat. No.
4,816,567). The "monoclonal antibodies" may also be isolated from phage
antibody libraries using the techniques described in Clackson et al.
(1991) Nature 352: 624-628 and Marks et al. (1991) J. Mol. Biol. 222:
581-597, for example.

[0030] The monoclonal antibodies herein specifically include "chimeric"
antibodies (immunoglobulins) in which a portion of the heavy and/or light
chain is identical with or homologous to corresponding sequences in
antibodies derived from a particular species or belonging to a particular
antibody class or subclass, while the remainder of the chain(s) is
identical with or homologous to corresponding sequences in antibodies
derived from another species or belonging to another antibody class or
subclass, as well as fragments of such antibodies, so long as they
exhibit the desired biological activity. U.S. Pat. No. 4,816,567;
Morrison et al. (1984) Proc. Natl. Acad. Sci. USA 81: 6851-6855.

[0031] A "domain antibody" is an immunologically functional immunoglobulin
fragment containing only the variable region of a heavy chain or the
variable region of a light chain. In some instances, two or more VH
regions are covalently joined with a peptide linker to create a bivalent
domain antibody. The two VH regions of a bivalent domain antibody
may target the same or different antigens.

[0032] A "bivalent antibody" comprises two antigen binding sites. In some
instances, the two binding sites have the same antigen specificities.
However, bivalent antibodies may be bispecific (see below).

[0033] As used herein, the term "single-chain Fv" or "scFv" antibody
refers to antibody fragments comprising the VH and VL domains
of antibody, wherein these domains are present in a single polypeptide
chain. Generally, the Fv polypeptide further comprises a polypeptide
linker between the VH and VL domains which enables the sFv to
form the desired structure for antigen binding. For a review of sFv, see
Pluckthun (1994) THE PHARMACOLOGY OF MONOCLONAL ANTIBODIES, vol. 113,
Rosenburg and Moore eds. Springer-Verlag, New York, pp. 269-315.

[0035] As used herein, the term "diabodies" refers to small antibody
fragments with two antigen-binding sites, which fragments comprise a
heavy chain variable domain (VH) connected to a light chain variable
domain (VL) in the same polypeptide chain (VH-VL or
VL-VH). By using a linker that is too short to allow pairing
between the two domains on the same chain, the domains are forced to pair
with the complementary domains of another chain and create two
antigen-binding sites. Diabodies are described more fully in, e.g., EP
404,097; WO 93/11161; and Holliger et al. (1993) Proc. Natl. Acad. Sci.
USA 90: 6444-6448. For a review of engineered antibody variants generally
see Holliger and Hudson (2005) Nat. Biotechnol. 23:1126-1136.

[0036] As used herein, the term "humanized antibody" refers to forms of
antibodies that contain sequences from non-human (e.g., murine)
antibodies as well as human antibodies. Such antibodies contain minimal
sequence derived from non-human immunoglobulin. In general, the humanized
antibody will comprise substantially all of at least one, and typically
two, variable domains, in which all or substantially all of the
hypervariable loops correspond to those of a non-human immunoglobulin and
all or substantially all of the FR regions are those of a human
immunoglobulin sequence. The humanized antibody optionally also will
comprise at least a portion of an immunoglobulin constant region (Fc),
typically that of a human immunoglobulin. The prefix "hum", "hu" or "h"
is added to antibody clone designations when necessary to distinguish
humanized antibodies from parental rodent antibodies. The humanized forms
of rodent antibodies will generally comprise the same CDR sequences of
the parental rodent antibodies, although certain amino acid substitutions
may be included to increase affinity, increase stability of the humanized
antibody, or for other reasons.

[0037] The antibodies of the present invention also include antibodies
with modified (or blocked) Fc regions to provide altered effector
functions. See, e.g., U.S. Pat. No. 5,624,821; WO2003/086310;
WO2005/120571; WO2006/0057702; Presta (2006) Adv. Drug Delivery Rev.
58:640-656. Such modification can be used to enhance or suppress various
reactions of the immune system, with possible beneficial effects in
diagnosis and therapy. Alterations of the Fc region include amino acid
changes (substitutions, deletions and insertions), glycosylation or
deglycosylation, and adding multiple Fc. Changes to the Fc can also alter
the half-life of antibodies in therapeutic antibodies, and a longer
half-life would result in less frequent dosing, with the concomitant
increased convenience and decreased use of material. See Presta (2005) J.
Allergy Clin. Immunol. 116:731 at 734-35.

[0038] The antibodies of the present invention also include antibodies
with intact Fc regions that provide full effector functions, e.g.
antibodies of isotype IgG1, which induce complement-dependent
cytotoxicity (CDC) or antibody dependent cellular cytotoxicity (ADCC) in
the a targeted cell.

[0039] The antibodies of the present invention also include antibodies
conjugated to cytotoxic payloads, such as cytotoxic agents or
radionuclides. Such antibody conjugates may be used in immunotherapy in
conjunction with anti-REG4 treatment, to selectively target and kill
cells expressing certain antigens on their surface. Exemplary cytotoxic
agents include ricin, vinca alkaloid, methotrexate, Psuedomonas exotoxin,
saporin, diphtheria toxin, cisplatin, doxorubicin, abrin toxin, gelonin
and pokeweed antiviral protein. Exemplary radionuclides for use in
immunotherapy with the antibodies of the present invention include
125I, 131I, 90Y, 67Cu, 211At, 177Lu,
143Pr and 213Bi. e.g., U.S. Patent Application Publication No.
2006/0014225.

[0040] The term "fully human antibody" refers to an antibody that
comprises human immunoglobulin protein sequences only. A fully human
antibody may contain murine carbohydrate chains if produced in a mouse,
in a mouse cell, or in a hybridoma derived from a mouse cell. Similarly,
"mouse antibody" or "rat antibody" refer to an antibody that comprises
only mouse or rat immunoglobulin sequences, respectively. A fully human
antibody may be generated in a human being, in a transgenic animal having
human immunoglobulin germline sequences, by phage display or other
molecular biological methods.

[0041] As used herein, the term "hypervariable region" refers to the amino
acid residues of an antibody that are responsible for antigen-binding.
The hypervariable region comprises amino acid residues from a
"complementarity determining region" or "CDR" (e.g. residues 24-34
(CDRL1), 50-56 (CDRL2) and 89-97 (CDRL3) in the light chain variable
domain and residues 31-35 (CDRH1), 50-65 (CDRH2) and 95-102 (CDRH3) in
the heavy chain variable domain (Kabat et al. (1991) Sequences of
Proteins of Immunological Interest, 5th Ed. Public Health Service,
National Institutes of Health, Bethesda, Md.) and/or those residues from
a "hypervariable loop" (i.e. residues 26-32 (L1), 50-52 (L2) and 91-96
(L3) in the light chain variable domain and 26-32 (H1), 53-55 (H2) and
96-101 (H3) in the heavy chain variable domain (Chothia and Lesk (1987)
J. Mol. Biol. 196: 901-917). As used herein, the term "framework" or "FR"
residues refers to those variable domain residues other than the
hypervariable region residues defined herein as CDR residues. The residue
numbering above relates to the Kabat numbering system and does not
necessarily correspond in detail to the sequence numbering in the
accompanying Sequence Listing.

[0042] "Binding compound" refers to a molecule, small molecule,
macromolecule, polypeptide, antibody or fragment or analogue thereof, or
soluble receptor, capable of binding to a target. "Binding compound" also
may refer to a complex of molecules, e.g., a non-covalent complex, to an
ionized molecule, and to a covalently or non-covalently modified
molecule, e.g., modified by phosphorylation, acylation, cross-linking,
cyclization, or limited cleavage, that is capable of binding to a target.
When used with reference to antibodies, the term "binding compound"
refers to both antibodies and antigen binding fragments thereof.
"Binding" refers to an association of the binding composition with a
target where the association results in reduction in the normal Brownian
motion of the binding composition, in cases where the binding composition
can be dissolved or suspended in solution. "Binding composition" refers
to a molecule, e.g. a binding compound, in combination with a stabilizer,
excipient, salt, buffer, solvent, or additive, capable of binding to a
target.

[0043] "Conservatively modified variants" or "conservative substitution"
refers to substitutions of amino acids are known to those of skill in
this art and may often be made even in essential regions of the
polypeptide without altering the biological activity of the resulting
molecule. Such exemplary substitutions are preferably made in accordance
with those set forth in Table 1 as follows:

[0044] Those of skill in this art recognize that, in general, single amino
acid substitutions in non-essential regions of a polypeptide may not
substantially alter biological activity. See, e.g., Watson et al. (1987)
Molecular Biology of the Gene, The Benjamin/Cummings Pub. Co., p. 224
(4th Edition).

[0045] The phrase "consists essentially of," or variations such as
"consist essentially of" or "consisting essentially of," as used
throughout the specification and claims, indicate the inclusion of any
recited elements or group of elements, and the optional inclusion of
other elements, of similar or different nature than the recited elements,
that do not materially change the basic or novel properties of the
specified dosage regimen, method, or composition. As a non-limiting
example, a binding compound that consists essentially of a recited amino
acid sequence may also include one or more amino acids, including
substitutions of one or more amino acid residues, that do not materially
affect the properties of the binding compound.

[0046] "Effective amount" encompasses an amount sufficient to ameliorate
or prevent a symptom or sign of the medical condition. Effective amount
also means an amount sufficient to allow or facilitate diagnosis. An
effective amount for a particular patient or veterinary subject may vary
depending on factors such as the condition being treated, the overall
health of the patient, the method route and dose of administration and
the severity of side affects. See, e.g., U.S. Pat. No. 5,888,530. An
effective amount can be the maximal dose or dosing protocol that avoids
significant side effects or toxic effects. The effect will result in an
improvement of a diagnostic measure or parameter by at least 5%, usually
by at least 10%, more usually at least 20%, most usually at least 30%,
preferably at least 40%, more preferably at least 50%, most preferably at
least 60%, ideally at least 70%, more ideally at least 80%, and most
ideally at least 90%, where 100% is defined as the diagnostic parameter
shown by a normal subject. See, e.g., Maynard et al. (1996) A Handbook of
SOPs for Good Clinical Practice, Interpharm Press, Boca Raton, Fla.; Dent
(2001) Good Laboratory and Good Clinical Practice, Urch Publ., London,
UK.

[0048] As cancerous cells grow and multiply, they form a mass of cancerous
tissue, that is a tumor, which invades and destroys normal adjacent
tissues. Malignant tumors are cancer. Malignant tumors usually can be
removed, but they may grow back. Cells from malignant tumors can invade
and damage nearby tissues and organs. Also, cancer cells can break away
from a malignant tumor and enter the bloodstream or lymphatic system,
which is the way cancer cells spread from the primary tumor (i.e., the
original cancer) to form new tumors in other organs. The spread of cancer
in the body is called metastasis (What You Need to Know About Cancer--an
Overview, NIH Publication No. 00-1566; posted Sep. 26, 2000, updated Sep.
16, 2002 (2002)).

[0049] As used herein, the term "solid tumor" refers to an abnormal growth
or mass of tissue that usually does not contain cysts or liquid areas.
Solid tumors may be benign (not cancerous) or malignant (cancerous).
Different types of solid tumors are named for the type of cells that form
them. Examples of solid tumors are sarcomas, carcinomas, and lymphomas.
Leukemias (cancers of the blood) generally do not form solid tumors
(National Cancer Institute, Dictionary of Cancer Terms).

[0050] As used herein, the term "primary cancer" refers to the original
tumor or the first tumor. Cancer may begin in any organ or tissue of the
body. It is usually named for the part of the body or the type of cell in
which it originates (Metastatic Cancer: Questions and Answers, Cancer
Facts 6.20, National Cancer Institute, reviewed Sep. 1, 2004 (2004)).

[0051] As used herein, the term "carcinoma in situ" refers to cancerous
cells that are still contained within the tissue where they started to
grow, and have not yet become invasive or spread to other parts of the
body.

[0052] As used herein, the term "carcinomas" refers to cancers of
epithelial cells, which are cells that cover the surface of the body,
produce hormones, and make up glands. Examples of carcinomas are cancers
of the skin, lung, colon, stomach, breast, prostate and thyroid gland.

[0053] As used herein, the term "isolated nucleic acid molecule" refers to
a nucleic acid molecule that is identified and separated from at least
one contaminant nucleic acid molecule with which it is ordinarily
associated in the natural source of the antibody nucleic acid. An
isolated nucleic acid molecule is other than in the form or setting in
which it is found in nature. Isolated nucleic acid molecules therefore
are distinguished from the nucleic acid molecule as it exists in natural
cells. However, an isolated nucleic acid molecule includes a nucleic acid
molecule contained in cells that ordinarily express the antibody where,
for example, the nucleic acid molecule is in a chromosomal location
different from that of natural cells.

[0054] The expression "control sequences" refers to DNA sequences involved
in the expression of an operably linked coding sequence in a particular
host organism. The control sequences that are suitable for prokaryotes,
for example, include a promoter, optionally an operator sequence, and a
ribosome binding site. Eukaryotic cells are known to use promoters,
polyadenylation signals, and enhancers.

[0055] A nucleic acid is "operably linked" when it is placed into a
functional relationship with another nucleic acid sequence. For example,
DNA for a presequence or secretory leader is operably linked to DNA for a
polypeptide if it is expressed as a preprotein that participates in the
secretion of the polypeptide; a promoter or enhancer is operably linked
to a coding sequence if it affects the transcription of the sequence; or
a ribosome binding site is operably linked to a coding sequence if it is
positioned so as to facilitate translation. Generally, "operably linked"
means that the DNA sequences being linked are contiguous, and, in the
case of a secretory leader, contiguous and in reading frame. However,
enhancers do not have to be contiguous. Linking is accomplished by
ligation at convenient restriction sites. If such sites do not exist, the
synthetic oligonucleotide adaptors or linkers are used in accordance with
conventional practice.

[0056] As used herein, the expressions "cell," "cell line," and "cell
culture" are used interchangeably and all such designations include
progeny. Thus, the words "transformants" and "transformed cells" include
the primary subject cell and cultures derived therefrom without regard
for the number of transfers. It is also understood that all progeny may
not be precisely identical in DNA content, due to deliberate or
inadvertent mutations. Mutant progeny that have the same function or
biological activity as screened for in the originally transformed cell
are included. Where distinct designations are intended, it will be clear
from the context.

[0057] As used herein, "polymerase chain reaction" or "PCR" refers to a
procedure or technique in which minute amounts of a specific piece of
nucleic acid, RNA and/or DNA, are amplified as described in, e.g., U.S.
Pat. No. 4,683,195. Generally, sequence information from the ends of the
region of interest or beyond needs to be available, such that
oligonucleotide primers can be designed; these primers will be identical
or similar in sequence to opposite strands of the template to be
amplified. The 5' terminal nucleotides of the two primers can coincide
with the ends of the amplified material. PCR can be used to amplify
specific RNA sequences, specific DNA sequences from total genomic DNA,
and cDNA transcribed from total cellular RNA, bacteriophage or plasmid
sequences, etc. See generally Mullis et al. (1987) Cold Spring Harbor
Symp. Quant. Biol. 51:263; Erlich, ed., (1989) PCR TECHNOLOGY (Stockton
Press, N.Y.) As used herein, PCR is considered to be one, but not the
only, example of a nucleic acid polymerase reaction method for amplifying
a nucleic acid test sample comprising the use of a known nucleic acid as
a primer and a nucleic acid polymerase to amplify or generate a specific
piece of nucleic acid.

[0058] As used herein, the term "germline sequence" refers to a sequence
of unrearranged immunoglobulin DNA sequences, including rodent (e.g.
mouse) and human germline sequences. Any suitable source of unrearranged
immunoglobulin DNA may be used. Human germline sequences may be obtained,
for example, from JOINSOLVER® germline databases on the website for
the National Institute of Arthritis and Musculoskeletal and Skin Diseases
of the United States National Institutes of Health. Mouse germline
sequences may be obtained, for example, as described in Giudicelli et al.
(2005) Nucleic Acids Res. 33:D256-D261.

[0059] To examine the extent of enhancement of REG4 activity, for example,
samples or assays comprising a given, e.g., protein, gene, cell, or
organism, are treated with a potential activating or inhibiting agent and
are compared to control samples without the agent. Control samples, i.e.,
not treated with agent, are assigned a relative activity value of 100%.
Inhibition is achieved when the activity value relative to the control is
about 90% or less, typically 85% or less, more typically 80% or less,
most typically 75% or less, generally 70% or less, more generally 65% or
less, most generally 60% or less, typically 55% or less, usually 50% or
less, more usually 45% or less, most usually 40% or less, preferably 35%
or less, more preferably 30% or less, still more preferably 25% or less,
and most preferably less than 20%. Activation is achieved when the
activity value relative to the control is about 110%, generally at least
120%, more generally at least 140%, more generally at least 160%, often
at least 180%, more often at least 2-fold, most often at least 2.5-fold,
usually at least 5-fold, more usually at least 10-fold, preferably at
least 20-fold, more preferably at least 40-fold, and most preferably over
40-fold higher.

[0061] An endpoint of inhibition is generally 75% of the control or less,
preferably 50% of the control or less, more preferably 25% of the control
or less, and most preferably 10% of the control or less. Generally, an
endpoint of activation is at least 150% the control, preferably at least
two times the control, more preferably at least four times the control,
and most preferably at least 10 times the control.

[0062] "Small molecule" is defined as a molecule with a molecular weight
that is less than 10 kDa, typically less than 2 kDa, and preferably less
than 1 kDa. Small molecules include, but are not limited to, inorganic
molecules, organic molecules, organic molecules containing an inorganic
component, molecules comprising a radioactive atom, synthetic molecules,
peptide mimetics, and antibody mimetics. As a therapeutic, a small
molecule may be more permeable to cells, less susceptible to degradation,
and less apt to elicit an immune response than large molecules. Small
molecules, such as peptide mimetics of antibodies and cytokines, as well
as small molecule toxins are described. See, e.g., Casset et al. (2003)
Biochem. Biophys. Res. Commun. 307:198-205; Muyldermans (2001) J.
Biotechnol. 74:277-302; Li (2000) Nat. Biotechnol. 18:1251-1256;
Apostolopoulos et al. (2002) Curr. Med. Chem. 9:411-420; Monfardini et
al. (2002) Curr. Pharm. Des. 8:2185-2199; Domingues et al. (1999) Nat.
Struct. Biol. 6:652-656; Sato and Sone (2003) Biochem. J. 371:603-608;
U.S. Pat. No. 6,326,482.

[0063] "Specifically" or "selectively" binds, when referring to a
ligand/receptor, antibody/antigen, or other binding pair, indicates a
binding reaction that is determinative of the presence of the protein in
a heterogeneous population of proteins and other biologics. Thus, under
designated conditions, a specified ligand binds to a particular receptor
and does not bind in a significant amount to other proteins present in
the sample. As used herein, an antibody is said to bind specifically to a
polypeptide comprising a given sequence (in this case REG4) if it binds
to polypeptides comprising the sequence of REG4 but does not bind to
proteins lacking the sequence of REG4. For example, an antibody that
specifically binds to a polypeptide comprising REG4 may bind to a
FLAG®-tagged form of REG4 but will not bind to other FLAG®-tagged
proteins.

[0064] The antibody, or binding composition derived from the
antigen-binding site of an antibody, of the contemplated method binds to
its antigen with an affinity that is at least two fold greater,
preferably at least ten times greater, more preferably at least 20-times
greater, and most preferably at least 100-times greater than the affinity
with unrelated antigens. In a preferred embodiment the antibody will have
an affinity that is greater than about 109 liters/mol, as
determined, e.g., by Scatchard analysis. Munsen et al. (1980) Analyt.
Biochem. 107:220-239.

General

[0065] The present invention provides engineered anti-REG4 antibodies and
uses thereof to treat proliferative disorders, in particular cancers and
tumors. The expression of REG4, as noted above, is upregulated in the
progression of proliferative diseases.

[0067] Using siRNA mediated knockdown to model the effects of neutralizing
REG4 activity, it was confirmed that expansion of cell numbers
(colorectal and prostate cancer lines) on plastic was inhibited. In
addition to the impact on proliferation, there was a demonstrated
inhibitory effect of REG4 knockdown in cell cycle. Conversely, an
enhancement of the growth of a cell line that does not make REG4
endogenously (HCT116), resulted when exogenous recombinant REG4 was
administered.

[0068] The proliferation findings were extended by demonstrating that REG4
protein knockdown inhibited anchorage-independent growth, as measured by
colony growth from single cells in soft-agar, as well as spheroid growth
in suspension. Soft agar growth and spheroid growth assays are more
relevant to cancer biology than growth on plastic. It was further
demonstrated that REG4 knockdown induces apoptosis in these cell lines
suggesting that neutralizing REG4 will not only be cytostatic for cancer
cells, but will cause tumor regression.

[0069] While the primary focus of these studies was on the growth and
survival of the tumor cell, studies to explore other cancer relevant
biologies were also undertaken. Consistent with a potential role in
angiogenesis, rhREG4 potentiated tubule formation in HUVECs in vitro,
suggesting that REG4 is not only important for the growth and survival of
tumor cells directly, but can also aid the tumor in recruiting a blood
supply.

[0070] In addition to studying the phenotypic consequences of neutralizing
REG4 in cancer cells, the mechanism of action by which REG4 might be
acting was also studied. It had been previously shown that REG4 treatment
or over-expression leads to increased phosphorylation of EGFR (see, e.g.,
Bishhupuri (2006) supra). The data of the present invention indicates the
opposite effect, that neutralization of REG4 in PC3 cells, leads to
decreased pEGFR, it was also observed that this effect was not universal,
but seemingly cell line specific. For example, in a different cancer line
(KM12), it was observed that REG4 knockdown led to decreased levels of
two other receptor tyrosine kinases (RTKs), phosphor-insulin receptor and
phosphor-IGF1R, but not pEGFR. Phosphor-insulin receptor and
phosphor-IGF1R in PC3 cells were downregulated. Data of the present
invention indicated that recombinant REG4 (mutated for stability) can
induce phosphorylation events of several RTKs, including IGF1R and IR, as
well as VEGFR and HGF receptor. Collectively, these observations indicate
that REG4 expression in cancers may lead to both autocrine and paracrine
transactivation of receptor tyrosine kinases that are cancer drivers. The
fact that that REG4 may drive phosphorylation events of multiple RTKs is
important in that it suggests that targeting, e.g., inhibiting, one
protein could impact multiple cancer-relevant pathways. This is distinct
from the information known publicly, which has only demonstrated an
impact on EGFR, a pathway for which there are already multiple
therapeutics in the clinic.

[0071] The impact of REG4 on phosphor-insulin receptor and phosphor-IGF1R
is important because both of these receptors are cancer drivers. However,
targeting the insulin receptor universally could have toxicity issues,
due to the widespread expression of this receptor. In contrast, by
targeting these RTKs indirectly through REG4, only those cells dependent
on REG4 will be affected, thus drastically impacting the potential
toxicity profile in a positive manner.

[0072] Among the challenges encountered in trying to design IGF1R-specific
inhibitors, i.e., inhibitors that do not bind to the highly homologous
insulin receptor, is that there is potential for decreased efficacy. The
present data suggest that targeting REG4 will be superior to targeting
the individual RTKs.

[0073] Consistent with additional previous observations, it was shown that
REG4 modulation (with knockdown of endogenous REG4 protein and treatment
with recombinant REG4 protein) resulted in modulation of pAkt and Bcl-2.
In contrast to published information, it was demonstrated that Bcl-2
levels were not modulated at the transcriptional level, but rather at the
protein level. The present data also shows that other proteins, not
previously associated with REG4 modulation, appear to be regulated by
REG4. These include c-Fos, E2F-1, cyclin D1, pERK1, pERK2, GSK3beta,
p-p70 S6 kinase, RSK1, MSK2 & HSP27. Furthermore, the present data
suggest that ERK inhibition results in decreased REG4 levels. Together,
these results suggest a hypothesis that REG4 is up-regulated in cancer to
allow survival, enhance growth and perhaps stimulate motility of cancer
cells, when these cells would otherwise respond to signals to die.

[0074] Lastly, it was observed that REG4 activity modulated HSP27,
suggesting an additional mechanism by which REG4 can mediate
chemo-resistance analogous to the modulation of DPYD previously observed
(see, e.g., Mitani, et al (2007) supra). Therefore, a REG4 antagonistic
therapeutic combined with either current or future chemotherapeutics,
biologics or immunotherapeutics, should result in increased tumor
regression.

[0075] Antibodies of the present invention demonstrated the ability to
inhibit exogenous REG4 induced proliferation f HCT116 cells. Additionally
these antibodies can inhibit growth of the PC3 cancer cell line that
endogenously expresses REG4. Combining these antibodies with small
molecule inhibitors of pathways downstream of REG4 increased the efficacy
of either the antibodies or the small molecule inhibitors alone.

Generation of REG4 Specific Antibodies

[0076] Any suitable method for generating monoclonal antibodies may be
used. For example, a recipient may be immunized with REG4 or a fragment
thereof. Any suitable method of immunization can be used. Such methods
can include adjuvants, other immunostimulants, repeated booster
immunizations, and the use of one or more immunization routes. Any
suitable source of REG4 can be used as the immunogen for the generation
of the non-human antibody of the compositions and methods disclosed
herein. Such forms include, but are not limited whole protein,
peptide(s), and epitopes generated through recombinant, synthetic,
chemical or enzymatic degradation means known in the art.

[0077] Any form of the antigen can be used to generate the antibody that
is sufficient to generate a biologically active antibody. Thus, the
eliciting antigen may be a single epitope, multiple epitopes, or the
entire protein alone or in combination with one or more immunogenicity
enhancing agents known in the art. The eliciting antigen may be an
isolated full-length protein, a cell surface protein (e.g., immunizing
with cells transfected with at least a portion of the antigen), or a
soluble protein (e.g., immunizing with only the extracellular domain
portion of the protein). The antigen may be produced in a genetically
modified cell. The DNA encoding the antigen may genomic or non-genomic
(e.g., cDNA) and encodes at least a portion of the extracellular domain.
As used herein, the term "portion" refers to the minimal number of amino
acids or nucleic acids, as appropriate, to constitute an immunogenic
epitope of the antigen of interest. Any genetic vectors suitable for
transformation of the cells of interest may be employed, including but
not limited to adenoviral vectors, plasmids, and non-viral vectors, such
as cationic lipids.

[0078] Any suitable method can be used to elicit an antibody with the
desired biologic properties to inhibit REG4 activity. It is desirable to
prepare monoclonal antibodies (mAbs) from various mammalian hosts, such
as mice, rats, other rodents, humans, other primates, etc. Description of
techniques for preparing such monoclonal antibodies may be found in,
e.g., Stites et al. (eds.) BASIC AND CLINICAL IMMUNOLOGY (4th ed.) Lange
Medical Publications, Los Altos, Calif., and references cited therein;
Harlow and Lane (1988) ANTIBODIES: A LABORATORY MANUAL CSH Press; Goding
(1986) MONOCLONAL ANTIBODIES: PRINCIPLES AND PRACTICE (2d ed.) Academic
Press, New York, N.Y. Thus, monoclonal antibodies may be obtained by a
variety of techniques familiar to researchers skilled in the art.
Typically, spleen cells from an animal immunized with a desired antigen
are immortalized, commonly by fusion with a myeloma cell. See Kohler and
Milstein (1976) Eur. J. Immunol. 6:511-519. Alternative methods of
immortalization include transformation with Epstein Barr Virus,
oncogenes, or retroviruses, or other methods known in the art. See, e.g.,
Doyle et al. (eds. 1994 and periodic supplements) CELL AND TISSUE
CULTURE: LABORATORY PROCEDURES, John Wiley and Sons, New York, N.Y.
Colonies arising from single immortalized cells are screened for
production of antibodies of the desired specificity and affinity for the
antigen, and yield of the monoclonal antibodies produced by such cells
may be enhanced by various techniques, including injection into the
peritoneal cavity of a vertebrate host. Alternatively, one may isolate
DNA sequences that encode a monoclonal antibody or a antigen binding
fragment thereof by screening a DNA library from human B cells according,
e.g., to the general protocol outlined by Huse et al. (1989) Science
246:1275-1281.

[0079] Other suitable techniques involve selection of libraries of
antibodies in phage or similar vectors. See, e.g., Huse et al. supra; and
Ward et al. (1989) Nature 341:544-546. The polypeptides and antibodies of
the present invention may be used with or without modification, including
chimeric or humanized antibodies. Frequently, the polypeptides and
antibodies will be labeled by joining, either covalently or
non-covalently, a substance that provides for a detectable signal. A wide
variety of labels and conjugation techniques are known and are reported
extensively in both the scientific and patent literature. Suitable labels
include radionuclides, enzymes, substrates, cofactors, inhibitors,
fluorescent moieties, chemiluminescent moieties, magnetic particles, and
the like. Patents teaching the use of such labels include U.S. Pat. Nos.
3,817,837; 3,850,752; 3,939,350; 3,996,345; 4,277,437; 4,275,149; and
4,366,241. Also, recombinant immunoglobulins may be produced, see Cabilly
U.S. Pat. No. 4,816,567; and Queen et al. (1989) Proc. Nat'l Acad. Sci.
USA 86:10029-10033; or made in transgenic mice, see Mendez et al. (1997)
Nature Genetics 15:146-156. See also Abgenix and Medarex technologies.

[0080] Antibodies or binding compositions against predetermined fragments
of REG4 can be raised by immunization of animals with conjugates of the
polypeptide, fragments, peptides, or epitopes with carrier proteins.
Monoclonal antibodies are prepared from cells secreting the desired
antibody. These antibodies can be screened for binding to normal or
defective REG4. These monoclonal antibodies will usually bind with at
least a Kd of about 1 μM, more usually at least about 300 nM, 30
nM, 10 nM, 3 nM, 1 nM, 300 pM, 100 pM, 30 pM or better, usually
determined by ELISA. Suitable non-human antibodies may also be identified
using the biologic assays described in Examples 5 and 6, below.

Humanization of REG4 Specific Antibodies

[0081] Any suitable non-human antibody can be used as a source for the
hypervariable region. Sources for non-human antibodies include, but are
not limited to, murine (e.g. Mus musculus), rat (e.g. Rattus norvegicus),
Lagomorphs (including rabbits), bovine, and primates. For the most part,
humanized antibodies are human immunoglobulins (recipient antibody) in
which hypervariable region residues of the recipient are replaced by
hypervariable region residues from a non-human species (donor antibody)
such as mouse, rat, rabbit or non-human primate having the desired
specificity, affinity, and capacity. In some instances, Fv framework
region (FR) residues of the human immunoglobulin are replaced by
corresponding non-human residues. Furthermore, humanized antibodies may
comprise residues that are not found in the recipient antibody or in the
donor antibody. These modifications are made to further refine antibody
performance of the desired biological activity. For further details, see
Jones et al. (1986) Nature 321:522-525; Reichmann et al. (1988) Nature
332:323-329; and Presta (1992) Curr. Op. Struct. Biol. 2:593-596.

[0083] Amino acid sequence variants of humanized anti-REG4 antibody are
prepared by introducing appropriate nucleotide changes into the humanized
anti-REG4 antibody DNA, or by peptide synthesis. Such variants include,
for example, deletions from, and/or insertions into, and/or substitutions
of, residues within the amino acid sequences shown for the humanized
anti-REG4 antibody. Any combination of deletion, insertion, and
substitution is made to arrive at the final construct, provided that the
final construct possesses the desired characteristics. The amino acid
changes also may alter post-translational processes of the humanized
anti-REG4 antibody, such as changing the number or position of
glycosylation sites.

[0084] A useful method for identification of certain residues or regions
of the humanized anti-REG4 antibody polypeptide that are preferred
locations for mutagenesis is called "alanine scanning mutagenesis," as
described by Cunningham and Wells (1989) Science 244: 1081-1085. Here, a
residue or group of target residues are identified (e.g., charged
residues such as Arg, Asp, His, Lys, and Glu) and replaced by a neutral
or negatively charged amino acid (most preferably alanine or polyalanine)
to affect the interaction of the amino acids with REG4 antigen. The amino
acid residues demonstrating functional sensitivity to the substitutions
then are refined by introducing further or other variants at, or for, the
sites of substitution. Thus, while the site for introducing an amino acid
sequence variation is predetermined, the nature of the mutation per se
need not be predetermined. For example, to analyze the performance of a
mutation at a given site, Ala scanning or random mutagenesis is conducted
at the target codon or region and the expressed humanized anti-REG4
antibody variants are screened for the desired activity.

[0085] Amino acid sequence insertions include amino- and/or
carboxyl-terminal fusions ranging in length from one residue to
polypeptides containing a hundred or more residues, as well as
intrasequence insertions of single or multiple amino acid residues.
Examples of terminal insertions include humanized anti-REG4 antibody with
an N-terminal methionyl residue or the antibody fused to an epitope tag.
Other insertional variants of the humanized anti-REG4 antibody molecule
include the fusion to the N- or C-terminus of humanized anti-REG4
antibody of an enzyme or a polypeptide that increases the serum half-life
of the antibody.

[0086] Another type of variant is an amino acid substitution variant.
These variants have at least one amino acid residue in the humanized
anti-REG4 antibody molecule removed and a different residue inserted in
its place. The sites of greatest interest for substitutional mutagenesis
include the hypervariable loops, but FR alterations are also
contemplated.

[0087] Another type of amino acid variant of the antibody alters the
original glycosylation pattern of the antibody. By altering is meant
deleting one or more carbohydrate moieties found in the antibody, and/or
adding one or more glycosylation sites that are not present in the
antibody. Glycosylation of antibodies is typically either N-linked or
O-linked. N-linked refers to the attachment of the carbohydrate moiety to
the side chain of an asparagine residue. The tripeptide sequences
asparagine-X-serine and asparagine-X-threonine, where X is any amino acid
except proline, are the recognition sequences for enzymatic attachment of
the carbohydrate moiety to the asparagine side chain. Thus, the presence
of either of these tripeptide sequences in a polypeptide creates a
potential glycosylation site. O-linked glycosylation refers to the
attachment of one of the sugars N-acetylgalactosamine, galactose, or
xylose to a hydroxyamino acid, most commonly serine or threonine,
although 5-hydroxyproline or 5-hydroxylysine may also be used.

[0088] Addition of glycosylation sites to the antibody is conveniently
accomplished by altering the amino acid sequence such that it contains
one or more of the above-described tripeptide sequences (for N-linked
glycosylation sites). The alteration may also be made by the addition of,
or substitution by, one or more serine or threonine residues to the
sequence of the original antibody (for O-linked glycosylation sites).

[0089] Yet another type of amino acid variant is the substitution of
residues to provide for greater chemical stability of the final humanized
antibody. For example, an asparagine (N) residue may be changed to reduce
the potential for formation of isoaspartate at any NG sequences within a
rodent CDR. A similar problem may occur at a DG sequence. Reissner and
Aswad (2003) Cell. Mol. Life Sci. 60:1281. Isoaspartate formation may
debilitate or completely abrogate binding of an antibody to its target
antigen. Presta (2005) J. Allergy Clin. Immunol. 116:731 at 734. In one
embodiment, the asparagine is changed to glutamine (Q). In addition,
methionine residues in rodent CDRs may be changed to reduce the
possibility that the methionine sulfur would oxidize, which could reduce
antigen binding affinity and also contribute to molecular heterogeneity
in the final antibody preparation. Id. In one embodiment, the methionine
is changed to alanine (A). Antibodies with such substitutions are
subsequently screened to ensure that the substitutions do not decrease
REG4 binding affinity to unacceptable levels.

[0090] Nucleic acid molecules encoding amino acid sequence variants of
humanized REG4 specific antibody are prepared by a variety of methods
known in the art. These methods include, but are not limited to,
isolation from a natural source (in the case of naturally occurring amino
acid sequence variants) or preparation by oligonucleotide-mediated (or
site-directed) mutagenesis, PCR mutagenesis, and cassette mutagenesis of
an earlier prepared variant or a non-variant version of humanized
anti-REG4 antibody.

[0091] Ordinarily, amino acid sequence variants of the humanized anti-REG4
antibody will have an amino acid sequence having at least 75% amino acid
sequence identity with the original humanized antibody amino acid
sequences of either the heavy or the light chain more preferably at least
80%, more preferably at least 85%, more preferably at least 90%, and most
preferably at least 95%, 98% or 99%. Identity or homology with respect to
this sequence is defined herein as the percentage of amino acid residues
in the candidate sequence that are identical with the humanized anti-REG4
residues, after aligning the sequences and introducing gaps, if
necessary, to achieve the maximum percent sequence identity, and not
considering any conservative substitutions as part of the sequence
identity. None of N-terminal, C-terminal, or internal extensions,
deletions, or insertions into the antibody sequence shall be construed as
affecting sequence identity or homology.

[0092] The humanized antibody can be selected from any class of
immunoglobulins, including IgM, IgG, IgD, IgA, and IgE. Preferably, the
antibody is an IgG antibody. Any isotype of IgG can be used, including
IgG1, IgG2, IgG3, and IgG4. Variants of the IgG
isotypes are also contemplated. The humanized antibody may comprise
sequences from more than one class or isotype. Optimization of the
necessary constant domain sequences to generate the desired biologic
activity is readily achieved by screening the antibodies in the
biological assays described in the Examples.

[0093] Likewise, either class of light chain can be used in the
compositions and methods herein. Specifically, kappa, lambda, or variants
thereof are useful in the present compositions and methods.

[0094] Any suitable portion of the CDR sequences from the non-human
antibody can be used. The CDR sequences can be mutagenized by
substitution, insertion or deletion of at least one residue such that the
CDR sequence is distinct from the human and non-human antibody sequence
employed. It is contemplated that such mutations would be minimal.
Typically, at least 75% of the humanized antibody residues will
correspond to those of the non-human CDR residues, more often 90%, and
most preferably greater than 95%.

[0095] Any suitable portion of the FR sequences from the human antibody
can be used. The FR sequences can be mutagenized by substitution,
insertion or deletion of at least one residue such that the FR sequence
is distinct from the human and non-human antibody sequence employed. It
is contemplated that such mutations would be minimal. Typically, at least
75% of the humanized antibody residues will correspond to those of the
human FR residues, more often 90%, and most preferably greater than 95%,
98% or 99%.

[0097] In one embodiment, CDRs include variants of any single sequence CDR
disclosed herein (SEQ ID NOs: 17-64), in which the variant comprises 1,
2, 3, 4, 5, 6, 7, 8, 9, or more conservative amino acid substitutions
relative to the disclosed sequence, as determined using the data of Table
1.

[0098] Also contemplated are chimeric antibodies. As noted above, typical
chimeric antibodies comprise a portion of the heavy and/or light chain
identical with, or homologous to, corresponding sequences in antibodies
derived from a particular species or belonging to a particular antibody
class or subclass, while the remainder of the chain(s) is identical with
or homologous to corresponding sequences in antibodies derived from
another species or belonging to another antibody class or subclass, as
well as fragments of such antibodies, so long as they exhibit the desired
biological activity. See U.S. Pat. No. 4,816,567; and Morrison et al.
(1984) Proc. Natl. Acad. Sci. USA 81: 6851-6855.

[0099] Bispecific antibodies are also useful in the present methods and
compositions. As used herein, the term "bispecific antibody" refers to an
antibody, typically a monoclonal antibody, having binding specificities
for at least two different antigenic epitopes. In one embodiment, the
epitopes are from the same antigen. In another embodiment, the epitopes
are from two different antigens. Methods for making bispecific antibodies
are known in the art. For example, bispecific antibodies can be produced
recombinantly using the co-expression of two immunoglobulin heavy
chain/light chain pairs. See, e.g., Milstein et al. (1983) Nature 305:
537-39. Alternatively, bispecific antibodies can be prepared using
chemical linkage. See, e.g., Brennan et al. (1985) Science 229:81.
Bispecific antibodies include bispecific antibody fragments. See, e.g.,
Holliger et al. (1993) Proc. Natl. Acad. Sci. U.S.A. 90:6444-48, Gruber
et al. (1994) J. Immunol. 152:5368.

[0100] In yet other embodiments, different constant domains may be
appended to humanized VL and VH regions derived from the CDRs
provided herein. For example, if a particular intended use of an antibody
(or fragment) of the present invention were to call for altered effector
functions, a heavy chain constant domain other than IgG1 may be used.
Although IgG1 antibodies provide for long half-life and for effector
functions, such as complement activation and antibody-dependent cellular
cytotoxicity, such activities may not be desirable for all uses of the
antibody. In such instances an IgG4 constant domain, for example, may be
used.

[0101] The parental and engineered forms of the antibodies of the present
invention may also be conjugated to a chemical moiety. The chemical
moiety may be, inter alia, a polymer, a radionuclide or a cytotoxic
factor. Preferably the chemical moiety is a polymer which increases the
half-life of the antibody molecule in the body of a subject. Suitable
polymers include, but are not limited to, polyethylene glycol (PEG)
(e.g., PEG with a molecular weight of 2 kDa, 5 kDa, 10 kDa, 12 kDa, 20
kDa, 30 kDa or 40 kDa), dextran and monomethoxypolyethylene glycol
(mPEG). Lee et al., (1999) (Bioconj. Chem. 10:973-981) discloses PEG
conjugated single-chain antibodies. Wen et al., (2001) (Bioconj. Chem.
12:545-553) disclose conjugating antibodies with PEG which is attached to
a radiometal chelator (diethylenetriaminpentaacetic acid (DTPA)).

[0103] The antibodies and antibody fragments or the REG4 soluble proteins
or fragments thereof of the invention may also be conjugated with
fluorescent or chemilluminescent labels, including fluorophores such as
rare earth chelates, fluorescein and its derivatives, rhodamine and its
derivatives, isothiocyanate, phycoerythrin, phycocyanin, allophycocyanin,
o-phthaladehyde, fluorescamine, 152Eu, dansyl, umbelliferone,
luciferin, luminal label, isoluminal label, an aromatic acridinium ester
label, an imidazole label, an acridimium salt label, an oxalate ester
label, an aequorin label, 2,3-dihydrophthalazinediones, biotin/avidin,
spin labels and stable free radicals.

[0104] Any method known in the art for conjugating the antibody molecules
or protein molecules of the invention to the various moieties may be
employed, including those methods described by Hunter et al., (1962)
Nature 144:945; David et al., (1974) Biochemistry 13:1014; Pain et al.,
(1981) J. Immunol. Meth. 40:219; and Nygren, J., (1982) Histochem. and
Cytochem. 30:407. Methods for conjugating antibodies and proteins are
conventional and very well known in the art.

Antibody Production

[0105] In one embodiment, for recombinant production of the antibodies of
the present invention, the nucleic acids encoding the two chains are
isolated and inserted into one or more replicable vectors for further
cloning (amplification of the DNA) or for expression. DNA encoding the
monoclonal antibody is readily isolated and sequenced using conventional
procedures (e.g., by using oligonucleotide probes that are capable of
binding specifically to genes encoding the heavy and light chains of the
antibody). Many vectors are available. The vector components generally
include, but are not limited to, one or more of the following: a signal
sequence, an origin of replication, one or more marker genes, an enhancer
element, a promoter, and a transcription termination sequence. In one
embodiment, both the light and heavy chains of a humanized anti-REG4
antibody of the present invention are expressed from the same vector,
e.g. a plasmid or an adenoviral vector.

[0106] Antibodies of the present invention may be produced by any method
known in the art. In one embodiment, antibodies are expressed in
mammalian or insect cells in culture, such as chinese hamster ovary (CHO)
cells, human embryonic kidney (HEK) 293 cells, mouse myeloma NSO cells,
baby hamster kidney (BHK) cells, Spodoptera frugiperda ovarian (Sf9)
cells. In one embodiment, antibodies secreted from CHO cells are
recovered and purified by standard chromatographic methods, such as
protein A, cation exchange, anion exchange, hydrophobic interaction, and
hydroxyapatite chromatography. Resulting antibodies are concentrated and
stored in 20 mM sodium acetate, pH 5.5.

[0107] In another embodiment, the antibodies of the present invention are
produced in yeast according to the methods described in WO2005/040395.
Briefly, vectors encoding the individual light or heavy chains of an
antibody of interest are introduced into different yeast haploid cells,
e.g. different mating types of the yeast Pichia pastoris, which yeast
haploid cells are optionally complementary auxotrophs. The transformed
haploid yeast cells can then be mated or fused to give a diploid yeast
cell capable of producing both the heavy and the light chains. The
diploid strain is then able to secret the fully assembled and
biologically active antibody. The relative expression levels of the two
chains can be optimized, for example, by using vectors with different
copy number, using transcriptional promoters of different strengths, or
inducing expression from inducible promoters driving transcription of the
genes encoding one or both chains.

[0108] In one embodiment, the respective heavy and light chains of a
plurality of different anti-REG4 antibodies (the "original" antibodies)
are introduced into yeast haploid cells to create a library of haploid
yeast strains of one mating type expressing a plurality of light chains,
and a library of haploid yeast strains of a different mating type
expressing a plurality of heavy chains. These libraries of haploid
strains can be mated (or fused as spheroplasts) to produce a series of
diploid yeast cells expressing a combinatorial library of antibodies
comprised of the various possible permutations of light and heavy chains.
The combinatorial library of antibodies can then be screened to determine
whether any of the antibodies has properties that are superior (e.g.
higher affinity for REG4) to those of the original antibodies. See. e.g.,
WO2005/040395.

[0109] In another embodiment, antibodies of the present invention are
human domain antibodies in which portions of an antibody variable domain
are linked in a polypeptide of molecular weight approximately 13 kDa.
See, e.g., U.S. Pat. Publication No. 2004/0110941. Such single domain,
low molecular weight agents provide numerous advantages in terms of ease
of synthesis, stability, and route of administration.

Biological Activity of Humanized Anti-REG4 Antibodies

[0110] Antibodies having the characteristics identified herein as being
desirable in a humanized anti-REG4 antibody can be screened for
inhibitory biologic activity in vitro or suitable binding affinity.
Agonist antibodies may be distinguished from antagonist antibodies using
the biological assay provided in the Examples below. Antibodies that
exhibit antagonist activity will block the activity of REG4.

[0111] To screen for antibodies that bind to the epitope on human REG4
bound by an antibody of interest (e.g., those that block binding of
REG4), a routine cross-blocking assay such as that described in
ANTIBODIES, A LABORATORY MANUAL, Cold Spring Harbor Laboratory, Ed Harlow
and David Lane (1988), can be performed. Antibodies that bind to the same
epitope are likely to cross-block in such assays, but not all
cross-blocking antibodies will necessarily bind at precisely the same
epitope since cross-blocking may result from steric hindrance of antibody
binding by antibodies bind at overlapping epitopes, or even nearby
non-overlapping epitopes.

[0112] Alternatively, epitope mapping, e.g., as described in Champe et al.
(1995) J. Biol. Chem. 270:1388-1394, can be performed to determine
whether the antibody binds an epitope of interest. "Alanine scanning
mutagenesis," as described by Cunningham and Wells (1989) Science 244:
1081-1085, or some other form of point mutagenesis of amino acid residues
in human REG4 may also be used to determine the functional epitope for an
anti-REG4 antibody of the present invention. Mutagenesis studies,
however, may also reveal amino acid residues that are crucial to the
overall three-dimensional structure of REG4 but that are not directly
involved in antibody-antigen contacts, and thus other methods may be
necessary to confirm a functional epitope determined using this method.

[0113] The epitope bound by a specific antibody may also be determined by
assessing binding of the antibody to peptides comprising fragments of
human REG4 (SEQ ID NO: 65). A series of overlapping peptides encompassing
the sequence of REG4 may be synthesized and screened for binding, e.g. in
a direct ELISA, a competitive ELISA (where the peptide is assessed for
its ability to prevent binding of an antibody to REG4 bound to a well of
a microtiter plate), or on a chip. Such peptide screening methods may not
be capable of detecting some discontinuous functional epitopes, i.e.
functional epitopes that involve amino acid residues that are not
contiguous along the primary sequence of the REG4 polypeptide chain.

[0114] The epitope bound by antibodies of the present invention may also
be determined by structural methods, such as X-ray crystal structure
determination (e.g., WO2005/044853), molecular modeling and nuclear
magnetic resonance (NMR) spectroscopy, including NMR determination of the
H-D exchange rates of labile amide hydrogens in REG4 when free and when
bound in a complex with an antibody of interest (Zinn-Justin et al.
(1992) Biochemistry 31:11335-11347; Zinn-Justin et al. (1993)
Biochemistry 32:6884-6891).

[0115] With regard to X-ray crystallography, crystallization may be
accomplished using any of the known methods in the art (e.g. Giege et al.
(1994) Acta Crystallogr. D50:339-350; McPherson (1990) Eur. J. Biochem.
189:1-23), including microbatch (e.g. Chayen (1997) Structure
5:1269-1274), hanging-drop vapor diffusion (e.g. McPherson (1976) J.
Biol. Chem. 251:6300-6303), seeding and dialysis. It is desirable to use
a protein preparation having a concentration of at least about 1 mg/mL
and preferably about 10 mg/mL to about 20 mg/mL. Crystallization may be
best achieved in a precipitant solution containing polyethylene glycol
1000-20,000 (PEG; average molecular weight ranging from about 1000 to
about 20,000 Da), preferably about 5000 to about 7000 Da, more preferably
about 6000 Da, with concentrations ranging from about 10% to about 30%
(w/v). It may also be desirable to include a protein stabilizing agent,
e.g. glycerol at a concentration ranging from about 0.5% to about 20%. A
suitable salt, such as sodium chloride, lithium chloride or sodium
citrate may also be desirable in the precipitant solution, preferably in
a concentration ranging from about 1 mM to about 1000 mM. The precipitant
is preferably buffered to a pH of from about 3.0 to about 5.0, preferably
about 4.0. Specific buffers useful in the precipitant solution may vary
and are well-known in the art. Scopes, Protein Purification: Principles
and Practice, Third ed., (1994) Springer-Verlag, New York. Examples of
useful buffers include, but are not limited to, HEPES, Tris, MES and
acetate. Crystals may be grow at a wide range of temperatures, including
2° C., 4° C., 8° C. and 26° C.

[0117] Additional antibodies binding to the same epitope as an antibody of
the present invention may be obtained, for example, by screening of
antibodies raised against REG4 for binding to the epitope, or by
immunization of an animal with a peptide comprising a fragment of human
REG4 comprising the epitope sequence. Antibodies that bind to the same
functional epitope might be expected to exhibit similar biological
activities, such as blocking receptor binding, and such activities can be
confirmed by functional assays of the antibodies.

[0118] Antibody affinities may be determined using standard analysis.
Preferred humanized antibodies are those that bind human REG4 with a
Kd value of no more than about 1×10-7; preferably no more
than about 1×10-8; more preferably no more than about
1×10-9; and most preferably no more than about
1×10-10 or even 1×10-11 M.

[0119] The antibodies and fragments thereof useful in the present
compositions and methods are biologically active antibodies and
fragments. As used herein, the term "biologically active" refers to an
antibody or antibody fragment that is capable of binding the desired the
antigenic epitope and directly or indirectly exerting a biologic effect.
As used herein, the term "specific" refers to the selective binding of
the antibody to the target antigen epitope. Antibodies can be tested for
specificity of binding by comparing binding to REG4 to binding to
irrelevant antigen or antigen mixture under a given set of conditions. If
the antibody binds to REG4 at least 10, and preferably 50 times more than
to irrelevant antigen or antigen mixture then it is considered to be
specific. An antibody that "specifically binds" to REG4 does not bind to
proteins that do not comprise the REG4-derived sequences, i.e.
"specificity" as used herein relates to REG4 specificity, and not any
other sequences that may be present in the protein in question. For
example, as used herein, an antibody that "specifically binds" to a
polypeptide comprising REG4 will typically bind to FLAG®-REG4, which
is a fusion protein comprising REG4 and a FLAG® peptide tag, but it
does not bind to the FLAG® peptide tag alone or when it is fused to a
protein other than REG4.

[0120] REG4-specific binding compounds of the present invention, such as
antagonistic REG4 specific antibodies, can inhibit its biological
activity in any manner, including but not limited to decreasing
proliferation of cells that respond to REG4. In particular, the REG4
binding compounds will inhibit phosphorylation events of various receptor
and intracellular molecules described above. The binding compounds can be
subjected to the biological assays provided herein.

[0123] Toxicity and therapeutic efficacy of the antibody compositions,
administered alone or in combination with an immunosuppressive agent, can
be determined by standard pharmaceutical procedures in cell cultures or
experimental animals, e.g., for determining the LD50 (the dose
lethal to 50% of the population) and the ED50 (the dose
therapeutically effective in 50% of the population). The dose ratio
between toxic and therapeutic effects is the therapeutic index and it can
be expressed as the ratio of LD50 to ED50. Antibodies
exhibiting high therapeutic indices are preferred. The data obtained from
these cell culture assays and animal studies can be used in formulating a
range of dosage for use in human. The dosage of such compounds lies
preferably within a range of circulating concentrations that include the
ED50 with little or no toxicity. The dosage may vary within this
range depending upon the dosage form employed and the route of
administration.

[0124] The mode of administration is not particularly important. Suitable
routes of administration may, for example, include oral, rectal,
transmucosal, or intestinal administration; parenteral delivery,
including intramuscular, subcutaneous, intramedullary injections, as well
as intrathecal, direct intraventricular, intravenous, intraperitoneal,
intranasal, or intraocular injections. Administration of antibody used in
the pharmaceutical composition or to practice the method of the present
invention can be carried out in a variety of conventional ways, such as
oral ingestion, inhalation, topical application or cutaneous,
subcutaneous, intraperitoneal, parenteral, intraarterial or intravenous
injection.

[0125] Alternately, one may administer the antibody in a local rather than
systemic manner, for example, via injection of the antibody directly into
an arthritic joint or pathogen-induced lesion characterized by
immunopathology, often in a depot or sustained release formulation.
Furthermore, one may administer the antibody in a targeted drug delivery
system, for example, in a liposome coated with a tissue-specific
antibody, targeting, for example, arthritic joint or pathogen-induced
lesion characterized by immunopathology. The liposomes will be targeted
to and taken up selectively by the afflicted tissue.

[0127] Determination of the appropriate dose is made by the clinician,
e.g., using parameters or factors known or suspected in the art to affect
treatment or predicted to affect treatment. Generally, the dose begins
with an amount somewhat less than the optimum dose and it is increased by
small increments thereafter until the desired or optimum effect is
achieved relative to any negative side effects. Important diagnostic
measures include those of symptoms of, e.g., the inflammation or level of
inflammatory cytokines produced. Preferably, a biologic that will be used
is substantially derived from the same species as the animal targeted for
treatment (e.g. a humanized antibody for treatment of human subjects),
thereby minimizing any immune response to the reagent.

[0129] As used herein, "inhibit" or "treat" or "treatment" includes a
postponement of development of the symptoms associated with autoimmune
disease or pathogen-induced immunopathology and/or a reduction in the
severity of such symptoms that will or are expected to develop. The terms
further include ameliorating existing uncontrolled or unwanted
autoimmune-related or pathogen-induced immunopathology symptoms,
preventing additional symptoms, and ameliorating or preventing the
underlying causes of such symptoms. Thus, the terms denote that a
beneficial result has been conferred on a vertebrate subject with an
autoimmune or pathogen-induced immunopathology disease or symptom, or
with the potential to develop such a disease or symptom.

[0130] As used herein, the term "therapeutically effective amount" or
"effective amount" refers to an amount of an REG4-specific binding
compound, e.g. and antibody, that when administered alone or in
combination with an additional therapeutic agent to a cell, tissue, or
subject is effective to prevent or ameliorate the autoimmune disease or
pathogen-induced immunopathology associated disease or condition or the
progression of the disease. A therapeutically effective dose further
refers to that amount of the compound sufficient to result in
amelioration of symptoms, e.g., treatment, healing, prevention or
amelioration of the relevant medical condition, or an increase in rate of
treatment, healing, prevention or amelioration of such conditions. When
applied to an individual active ingredient administered alone, a
therapeutically effective dose refers to that ingredient alone. When
applied to a combination, a therapeutically effective dose refers to
combined amounts of the active ingredients that result in the therapeutic
effect, whether administered in combination, serially or simultaneously.
An effective amount of therapeutic will decrease the symptoms typically
by at least 10%; usually by at least 20%; preferably at least about 30%;
more preferably at least 40%, and most preferably by at least 50%.

[0135] The present invention provides methods for using anti-REG4
antibodies and fragments thereof for the treatment and diagnosis of
proliferative disorders and conditions.

[0136] The present invention provides methods for diagnosing the presence
of a cancer by analyzing expression levels of REG4 in test cells, tissue
or bodily fluids compared with REG4 levels in cells, tissues or bodily
fluids of preferably the same type from a control. As demonstrated
herein, an increase in level of REG4 expression, for example, in the
patient versus the control is associated with the presence of cancer.

[0137] Typically, for a quantitative diagnostic assay, a positive result
indicating the patient tested has cancer or an infectious disease, is one
in which the cells, tissues, or bodily fluids has an REG4 expression
level at least two times higher, five times higher, ten times higher,
fifteen times higher, twenty times higher, twenty-five times higher.

[0138] Assay techniques that may be used to determine levels of gene and
protein expression, such as REG4, of the present inventions, in a sample
derived from a host are well known to those of skill in the art. Such
assay methods include radioimmunoassays, reverse transcriptase PCR
(RT-PCR) assays, quantitative real-time PCR assays, immunohistochemistry
assays, in situ hybridization assays, competitive-binding assays, western
blot assays, ELISA assays, and flow cytometric assays, for example, two
color FACS analysis for M2 versus M1 phenotyping of tumor-associated
macrophages (Mantovani et al., (2002) TRENDS in Immunology 23:549-555).

[0139] An ELISA assay initially comprises preparing an antibodies of the
present invention, specific to REG4, preferably 3C2.3D10, 4C5.3B10,
9F3.3A4, 12E1.3C11, 13E1.1B11, 40F6.3F6, 70A9.3C2, and 86C1.2B7
(collectively "REG4 antibodies"). In addition, a reporter antibody
generally is prepared that binds specifically to REG4. The reporter
antibody is attached to a detectable reagent such as radioactive,
fluorescent or an enzymatic reagent, for example horseradish peroxidase
enzyme or alkaline phosphatase.

[0140] To carry out the ELISA, at least one of the REG4 antibodies
described above is incubated on a solid support, e.g., a polystyrene dish
that binds the antibody. Any free protein binding sites on the dish are
then covered by incubating with a non-specific protein, such as bovine
serum albumin. Next, the sample to be analyzed is incubated in the dish,
during which time REG4 binds to the specific REG4 antibody attached to
the polystyrene dish. Unbound sample is washed out with buffer. A
reporter antibody specifically directed to REG4 and linked to horseradish
peroxidase is placed in the dish resulting in binding of the reporter
antibody to any monoclonal antibody bound to REG4. Unattached reporter
antibody is then washed out. Reagents for peroxidase activity, including
a calorimetric substrate are then added to the dish. Immobilized
peroxidase, linked to REG4 antibodies, produces a colored reaction
product. The amount of color developed in a given time period is
proportional to the amount of REG4 protein present in the sample.
Quantitative results typically are obtained by reference to a standard
curve.

[0141] A competition assay may be employed wherein antibodies specific to
REG4 are attached to a solid support and labeled REG4 and a sample
derived from the host are passed over the solid support and the amount of
label detected attached to the solid support can be correlated to a
quantity of REG4 in the sample.

[0142] The above tests may be carried out on samples derived from a
variety of cells, bodily fluids and/or tissue extracts such as
homogenates or solubilized tissue obtained from a patient. Tissue
extracts are obtained routinely from tissue biopsy and autopsy material.
Bodily fluids useful in the present invention include blood, urine,
saliva or any other bodily secretion or derivative thereof. The term
"blood" is meant to include whole blood, plasma, serum or any derivative
of blood.

[0143] The broad scope of this invention is best understood with reference
to the following examples, which are not intended to limit the inventions
to the specific embodiments. The specific embodiments described herein
are offered by way of example only, and the invention is to be limited by
the terms of the appended claims, along with the full scope of
equivalents to which such claims are entitled.

[0149] The humanization of antibodies is described generally, e.g., in PCT
patent application publications WO 2005/047324 and WO 2005/047326.

[0150] Briefly, the amino acid sequence of the non-human VH domain (e.g.
SEQ ID NOs: 1-8) is compared to a group of five human VH germline amino
acid sequences; one representative from subgroups IGHV1 and IGHV4 and
three representatives from subgroup IGHV3. The VH subgroups are listed in
M.-P. Lefranc (2001) "Nomenclature of the Human Immunoglobulin Heavy
(IGH) Genes", Experimental and Clinical Immunogenetics 18:100-116. The
framework sequences of the human germline sequence with the closest match
are used to construct a humanized VH domain.

[0151] The rodent anti-huREG4 antibodies disclosed herein are all of the
kappa subclass of VL. The amino acid sequences of the non-human VL domain
(e.g. SEQ ID NOs: 9-16) is compared to a group of four human VL kappa
germline amino acid sequences. The group of four is comprised of one
representative from each of four established human VL subgroups listed in
V. Barbie & M.-P. Lefranc (1998) "The Human Immunoglobulin Kappa Variable
(IGKV) Genes and Joining (IGKJ) Segments", Experimental and Clinical
Immunogenetics 15:171-183 and M.-P. Lefranc (2001) "Nomenclature of the
Human Immunoglobulin Kappa (IGK) Genes", Experimental and Clinical
Immunogenetics 18:161-174. The four subgroups also correspond to the four
subgroups listed in Kabat et al. (1991-5th Ed.) "Sequences of Proteins of
Immunological Interest", U.S. Department of Health and Human Services,
NIH Pub. 91-3242, pp. 103-130. The framework sequences of the human
germline sequence with the closest match are used to construct a
humanized VL domain.

[0152] Once the target amino acid sequences of the variable heavy and
light chains are determined, plasmids encoding the full-length humanized
antibody may be generated. Plasmid sequences may be altered using Kunkel
mutagenesis (see, e.g., Kunkel T A. (1985) Proc. Natl. Acad. Sci. U.S.A
82:488-492) to change the DNA sequence to the target humanized antibody
sequences. Simultaneously, codon optimization may be performed to provide
for potentially optimal expression.

[0153] Antibodies of the present invention can be humanized using a method
that identifies an acceptor germline sequence for a humanized antibody,
and comprises the steps of: a) identifying a non-human antibody that has
the desired biological activity; b) determining the amino acid sequence
of a non-human antibody VH and VL domains; and c) comparing the
nonhuman antibody sequence to a group of human germline sequences,
wherein the comparison comprises the substeps of: 1) assigning the
non-human V sequences residue numbers according to Kabat supra; 2)
delineating the CDR and FR regions in the sequence according to Kabat
supra; 3) assigning a predetermined numerical score at specific residue
position for which the non-human and human antibody germline sequences
are identical; and 4) totaling all of the residue scores to generate a
total score for each human germline sequence; and d) identifying the
human germline sequence with the highest total residue score as the
acceptor germline sequence. In one embodiment, the method further
comprises the substeps of: 5) assigning a numerical score of 1 for each
FR residue position for which the non-human and human antibody germline
sequences are identical that was not scored in substep (3) to germline
sequences with identical total residue scores after substep (4); 6)
totaling all of the residue scores to generate a total score for each
human germline sequence. In a specific embodiment, the non-human antibody
is specific for REG4 and enhances the biological activity of REG4. Also
provided herein is an antibody generated by the above method.

[0154] In one embodiment, the REG4 antibody is humanized using the
following method. First, the non-human VL and VH domains of the
REG4 antibody are cloned and sequenced, and the amino acid sequence
determined. Then, the non-human VH sequence are compared to a group
of three human VH germline amino acid sequences. The three groups
contain one representative from each of subgroups IGHV1, IGHV3 and IGHV4.
The VH subgroups are listed in M.-P. Lefranc, Exp. Clin.
Immunogenetics, 18:100-116 (2001). Specifically, the comparison with the
three germline sequences begins with the assignment of residue numbers to
the non-human VH sequence according to the Kabat numbering system.
See Kabat, et al., U.S. Department of Health and Human Services, NIH Pub.
91-3242 (5th Ed., 1991). The non-human VH sequence are then aligned
with each of the three human germline sequences. Since the V genes only
comprise VH residues 1-94, only these residues are considered in the
alignment. Next, the complementarity-determining (CDR) and framework (FR)
regions in the sequence are delineated. CDR and FR are delineated
according to the combination of the definitions provided in Kabat, et
al., U.S. Department of Health and Human Services, NIH Pub. 91-3242 (5th
Ed., 1991), and C. Chothia & A. M. Lesk, J. Mol. Biol., 196:901-917
(1987). Therefore, the CDR definition used is residues 26-35 for CDR1,
residues 50-65 for CDR2, and CDR3 is residues 95-102 for CDR3 of the
VH domain. The next step involves assigning a numerical score at
identified residue position where the non-human and human sequences are
identical. One example of this scoring is shown in Table 4 below.

[0155] After the residue positions are assigned a numerical score, all of
the residue scores are totaled. The acceptor germline sequence is the one
with the highest total score. In a case where two or more germline
sequences have identical scores, then add 1 to the total for each
position where the non-human and human sequences are IDENTICAL for the
following FR residues: 1-23, 25, 36, 38-47, 66, 68, 70, 72, 74, 75, 77,
and 79-93 (max 60). The residue scores are totaled again, and the
acceptor germline sequence is the one with the highest total score. If
two or more germline sequences still have identical scores, either one
can be used as the acceptor germline sequence.

[0156] If the VL sequence is a member of the kappa subclass of
VL, the non-human VL sequence from the REG4 specific antibody
is compared to a group of four human VL kappa germline amino acid
sequences. The four sequences are comprised of one representative from
each of four established human VL subgroups listed in V. Barbie &
M.-P. Lefranc, Exp. Clin. Immunogenetics 15:171-183 (1998) and M.-P.
Lefranc, Exp. Clin. Immunogenetics 18:161-174 (2001). The four sequences
also correspond to the four subgroups listed in Kabat et al., U.S.
Department of Health and Human Services, NIH Pub. 91-3242, pp. 103-130
(5th Ed., 1991). The comparison of the non-human sequence to the four
germline sequences begins with the assignment of residue numbers to the
non-human VL sequence residues according to Kabat et al., U.S.
Department of Health and Human Services, NIH Pub. 91-3242 (5th Ed.,
1991). The non-human VL sequences are then aligned with each of the
four human germline sequences. Since the V genes only comprise VL
residues 1-95, only these residues are considered in the alignment. Next,
the complementarity-determining (CDR) and framework (FR) regions are
delineated in the sequence. CDR and FR are delineated according to the
combination of the definitions provided in Kabat et al., U.S. Department
of Health and Human Services, NIH Pub. 91-3242 (5th Ed. 1991), and C.
Chothia & A. M. Lesk, J. Mol. Biol., 196:901-917 (1987). Therefore, the
CDR definition used is residues 24-34 for CDR1, residues 50-56 for CDR2,
and residues 89-97 for CDR3 of the VL domain. The next step involves
assigning a numerical score at identified residue position where the
non-human and human sequences are identical. One example of this scoring
is shown in Table 5 below.

[0157] After the residue positions are assigned a numerical score, all of
the residue scores are totaled. The acceptor germline sequence is the one
with the highest total score. In a case where two or more germline
sequences have identical scores, then add 1 to the total for each
position where the non-human and human sequences are IDENTICAL for the
following FR residues: 1-3, 5-23, 35-42, 44-49, 57, 59-88 (max 67). The
residue scores are totaled again, and the acceptor germline sequence is
the one with the highest total score. If two or more germline sequences
still have identical scores, either one can be used as the acceptor
germline sequence.

[0158] The equilibrium dissociation constants (Kd) for anti human
REG4 antibodies are determined using the KinExA 3000 instrument. Sapidyne
Instruments Inc., Boise Id., USA. KinExA uses the principle of the
Kinetic Exclusion Assay method based on measuring the concentration of
uncomplexed antibody in a mixture of antibody, antigen and
antibody-antigen complex. The concentration of free antibody is measured
by exposing the mixture to a solid-phase immobilized antigen for a very
brief period of time. In practice, this is accomplished by flowing the
solution phase antigen-antibody mixture past antigen-coated particles
trapped in a flow cell. Data generated by the instrument are analyzed
using custom software. Equilibrium constants are calculated using a
mathematical theory based on the following assumptions:

[0162] PMMA particles (Sapidyne, Cat No. 440198) are coated with
biotinylated REG4 (or a fragment thereof, such as the extracellular
domain) according to Sapidyne "Protocol for coating PMMA particles with
biotinylated ligands having short or nonexistent linker arms." EZ-link
TFP PEO-biotin (Pierce, Cat. No. 21219) is used for biotinylation of
REG4, as per the manufacturer's recommendations (Pierce bulletin 0874).

[0163] BIAcore determinations are performed essentially as described at
Example 4 of co-pending, commonly assigned U.S. patent application Ser.
No. 11/511,635 (filed 29 Aug. 2006). Briefly, ligands (anti-REG4-1-hIg)
are immobilized on a BIAcore CM5 sensor chip using standard
amine-coupling procedure. Kinetic constants for the various interactions
are determined using BIAevaluation software 3.1. The Kd is
determined using the calculated dissociation and association rate
constants.

[0164] The kinetic binding activity of Rat Anti-REG4_H antibodies against
Schering-Plough BioPharma REG4_H, lot 04ABY, was measured by surface
plasmon resonance using a Biacore T100 system (Biacore, GE Healthcare,
Piscataway, N.J.). Approximately 30 RUs of REG4 was immobilized via amine
coupling chemistry onto a Series S Sensor Chip CM5 (Research grade,
BR-1006-68). HBS-EP+ buffer (BR-1006-69) was used as the running buffer
with a flow rate of 304/min. Schering-Plough BioPharma Rat Anti-REG4_H
antibodies were injected over the immobilized REG4_H surface at varying
concentrations, ranging from [0.091 nM to 600 nM], at a flow rate of 30
μL/min. Following each injection cycle, the CM5 chip surface was
regenerated using a 10 mM Glycine pH 1.5 solution followed by an
injection of 25 mM NaOH solution at a flow rate of 75 μL/min.

[0169] Twenty-four hours after transfection, 3×103 cells were
plated in black view plates and incubated at 37° C. in a
humidified atmosphere with 5% CO2. Cell Titer Glo reagent (Promega G7573)
was added according to the manufacturer's directions 72-96 h
post-transfection. Briefly, 100 uL/well of Cell Titer Glo reagent was
added and the contents were mixed on the plate shaker at 400 rpm for 2
minutes to induce lysis. After a 10 minute incubation, the luminescence
was read with a multiwell plate reader (DTX 880 multimode reader) and
integration time of 100,000 μs, using the method of fluorescence
intensity.

Example 8

MTT Cell Proliferation Assay

[0170] At the end of assay, 10 ul MTT assay reagent (Roche, 11 465 007
001) was added to wells according to the manufacturer's directions. Four
hours later, 100 ul of solubilization solution was added to plates and
plates were incubated overnight in 37° C. incubator in a
humidified atmosphere. The absorbance of A550 nm-A690 nm was measured
with an ELISA plate reader (Molecular Devices Spectra max 340PC).

Example 9

Soft Agar Colony Formation Assay

[0171] 96-well flat bottom plates (Costar 3474) were coated with 1%
agarose (Cambrex 50070). Twenty-four hours after transfection, 5 to
10×102 cells/well were added in media with a final
concentration of 0.33% agarose. Wells were coated with 100 μL media
upon agarose solidification. After incubation for 7 days at 37° C.
in a humidified atmosphere with 5% CO2, 29 μL of Alamar blue
(Biosource DAL 1100) was added to each well. The fluorescence was read 4,
8, and 24 hours after the addition of Alamar blue using a multiwell plate
reader and a Rhodamine filter excitation 530 nm and emission 590 nm.

Example 10

Spheroid Assay

[0172] Round bottom 96-well plates were coated with Polyhema (Sigma
P3932). Twenty-four hours after transfection, cells were plated at a
density of 1×104 cells/well in media. Cells were grown on a
waver platform to allow spheroid formation at 37° C. in a
humidified atmosphere with 5% CO2 for 7 days. The supernatant was
transferred to a black assay plate and the spheroids lysed in a final
concentration of 2% Triton X-100. The Promega LDH kit (G7890) was used
according to the manufacturer's directions for the supernatant and lysed
spheroids to measure cell death and intracellular LDH from spheroids,
respectively. Briefly, 100 μL LDH reagent was added, shaked 30 seconds
and incubate 10 minutes before reading fluorescence on a plate reader at
560/590 nm.

Example 11

Cell Cycle Analysis by BrdU Incorporation

[0173] Cell cycle analysis was determined by transfecting cells with siRNA
for 24-72 h and harvesting (adherent and suspension) cells after being
pulsed for 30 minutes with 10 μM BrdU. Cells were fixed in 70% ethanol
and stored at -20° C. overnight. Cells were centrifuged,
resuspended in 0.1M HCl in PBS-0.5% Triton X-100 and incubated on ice for
10 minutes. Cells were washed with dH20, resuspended in dH20
and boiled for 10 minutes, followed by 10 minutes on ice. Cells were then
washed with PBS-0.5% Triton X-100 and resuspended in 10 uL 0.1% BSA, 20
uL anti-BrdU-FITC (BD 347583) and 70 uL PBS. After 30 minutes, cells were
washed and resuspended in propidium iodide-RNase solution (BD Pharmingen
550825). The cells were then analyzed for cell cycle perturbation using a
FACSCanto (Becton Dickinson). The FlowJo program was used to quantitate
the distribution of cells in each cell cycle phase: sub-G1
(apoptotic cells), G1, S, and G2-M.

[0174] Cell death was assessed by transfecting KM12 and PC3 cells plated
at a density of 1×105 cells/6-well plate with REG4 siRNA for
96 h and then stained with Annexin V-Alexa Fluor 488 (Invitrogen A13201)
as described in the manufacturer's protocol. Briefly, floating and
adherent cells were collected using cell dissociation buffer (Invitrogen
13151) and cells were incubated in 85 μL Annexin V binding buffer (BD
Pharmingen 556565), 5 μL Annexin V-Alexa Fluor 488 and 10 μL
propidium iodide (BD Pharmingen 550825) at 37° C. in the dark.
After 15 minutes, 400 μL of Annexin V binding buffer was added and
samples were analyzed within 30 minutes by flow cytometry using a
FACSCanto. FACS plots were analyzed in FlowJo to differentiate between
apoptotic and necrotic cells based on Annexin V and PI staining.

Example 13

Detection of Single Strand Breaks by F7-26 Staining

[0175] Detection of apoptosis was determined by transfecting KM12 and PC3
cells plated at a density of 1×105 cells/6-well plate with
REG4 siRNA for 96 h and then floating and adherent cells were collected
using 0.05% trypsin (Sigma 59417C). Cells were fixed in 100% methanol
overnight. Cells were then resuspended in 250 uL formamide and incubated
at 75 degrees for 15 minutes. Cells were blocked for 15 minutes in 1%
milk in PBS, probed with 1:10 dilution of F7-26 antibody (Chemicon
International MAB3299) for 15 minutes. Samples were then washed with PBS
and probed for 15 minutes with a 1:50 dilution of anti-mouse IgM
(Chemicon International AP128F) for 15 minutes in the dark. Cells were
resuspended in PI/RNase solution and analyzed using a FACSCanto. FACS
plots were analyzed in FlowJo to differentiate between normal and
apoptotic cells based on F7-26 staining.

[0178] Media was collected 48 h after transfection and centrifuged to
remove cell debris. Reg IV was immunoprecipitated from 2 mL samples with
Protein A/G beads, 40 ug mouse anti-Reg IV (R&D) or isotype control and
overnight incubation. IP samples were run by Western blot, as described
above.

Example 15

Exogenous REG4 Induced Growth/REG4 Antibody Screening

[0179] 96-well MaxiSorp plate (Nunc 12-565-136) were sterilized under UV
for 1 hour. Plates were coated with recombinant REG4 purchased from R&D
System (1379-RG-50) or produced in house at 1-20 μg/ml in 50 μl
DMEM medium at room temperature overnight in a moisturized chamber.
7×103HCT116 cells in 50 ul DMEM medium with 1% FBS were plated
to wells with and without 10-20 μg/ml anti-REG4 antibodies (e.g.,
3C2.3D10, 4C5.3B10, 9F3.3A4, 12E1.3C11, 13E1.1B11, 40F6.3F6, 70A9.3C2,
and 86C1.2B7) to a final 0.5% FBS concentration. MTT cell proliferation
assays were performed after cells had been incubated in 37° C.
tissue culture incubator for 45-48 hr.

Example 16

Anti-REG4 Activity in Inhibition of Endogenous REG4

[0180] PC3 and KM12 cells were transfection stressed for 24 h with 50 nM
pooled negative control siRNA from Invitrogen. Cells were re-plated in 96
well plates (BD 3072) at 1000 cells per well. REG4 antibodies were added
at a final concentration of 5-50 μg/ml in 1% FBS RPMI and plates were
incubated at 37° C. in a humidified chamber with 5% CO2 for 5
days with an exchange of 10 μg/well antibodies 48 h after prior
antibody treatment. On day 5, a MTT assay was performed according to the
kit directions.

Example 17

Exogenous Reg4 Induced MAPK Pathway Gene Activation

[0181] HCT116 cells plated overnight in tissue culture dishes were washed
once with serum free DMEM and serum starved in serum free DMEM for 24 h
at 37° C. in a humidified chamber with 5% CO2. 500 nM
recombinant Reg4 expressed in house in serum free DMEM was used to
stimulate the cells by incubation at 37° C. in a humidified
chamber with 5% CO2 for 5 minutes, 30 minutes and 2 hours. At the
end of stimulation, cells were washed once with PBS and lysed with RIPA
buffer (Sigma) containing phosphatase and protease inhibitors according
to the manufacturer's directions. 200 ug of cell lysate was applied to
MAPK arrays (R&D System, ARY002) and followed by incubation of detection
antibody cocktail and Streptavidin-HRP as recommended by manufacture.
Phospho-MAP kinase signals were detected on X-ray film after arrays were
exposed to chemiluminescent reagent (PIERCE, SuperSignal WestPico).

Example 18

Exogenous REG4 Induction of RTK Activation

[0182] HCT116 cells plated overnight in tissue culture dishes were washed
once with serum free DMEM and serum starved in serum free DMEM for 24 h
at 37° C. in a humidified chamber with 5% CO2. 500 nM
recombinant REG4 expressed in house in serum free DMEM was used to
stimulate the cells by incubation at 37° C. in a humidified
chamber with 5% CO2 for 5 minutes, 30 minutes and 2 hours. At the
end of stimulation, cells were washed once with PBS and lysed with RIPA
buffer (Sigma) containing phosphatase and protease inhibitors according
to the manufacturer's directions. 300 ug of cell lysate was applied to
RTK arrays (R&D System, ARY001) and followed by incubation of detection
antibody as recommended by manufacture. Phospho-RTK signals were detected
on X-ray film after arrays were exposed to chemiluminescent reagent
(PIERCE, SuperSignal WestPico).

Example 19

Exogenous REG4 Induction of p-Akt

Discovery-1 Detection

[0183] HCT116 cells were plated on collagen pre-coated 96-well black
plates (Sigma, S3190-5EA) and incubated overnight at 37° C. in a
humidified chamber with 5% CO2. Cells were washed once with serum
free DMEM and serum starved in serum free DMEM for 24 hours. 100 nM to
500 nM recombinant REG4 expressed in serum free DMEM was used to
stimulate the cells by incubation at 37° C. in a humidified
chamber with 5% CO2 for 5 minutes, 30 minutes and 2 hours. At the
end of stimulation, cells were fixed in 3% paraformaldehyde (Alfa Acesar,
30525-89-4) at room temperature for 15 minutes.

[0184] After one wash with PBS, cells were permeabilized in cold methanol
for 10 minutes in -20° C. and blocked with 3% BSA, 0.3% Triton
X-100 in PBS at room temperature for 1 hour. Cells then were incubated
with phospho-Akt antibody (S473; Cell Signaling Tech, #4060) diluted in
blocking buffer overnight at 4° C. After 3 washes by soaking the
cells in PBS for 10 minutes each, the secondary antibody, Alexa 488-Goat
anti-rabbit (Invitrogen) is added at a dilution of 1 to 500 in blocking
buffer and the cells were incubated at room temperature in dark for 2
hours. Cells were washed 3 times again by soaking in PBS for 10 minutes
each, followed by propidium iodide staining with Propidium Iodide/RNase A
solution (Becton-Dickenson) at room temperature for 10 minutes. Stained
cells were covered with DABCO mounting medium (Sigma, D2522) to prevent
cell lose and color diffusion. Fluorescence image capture and intensity
detection was acquired by using Discovery-1 detection system (Molecular
Devices) and images were analyzed with a journal written with version 6.1
Discovery-1 software with help from Molecular Devices technique support.
Phospho-Akt green fluorescence intensity induced by Reg4 was compared to
that of untreated cells after normalized with nuclei counts (fluorescence
in red) in the images captured.

Example 20

Exogenous REG4 Induction of p-Akt

MSD Detection

[0185] HCT116 cells plated overnight in tissue culture dishes were washed
once with serum free DMEM and serum starved in serum free DMEM for 24 h
at 37° C. in a humidified chamber with 5% CO2. 100 nM to 500
nM recombinant Reg4 expressed in house in serum free DMEM was used to
stimulate the cells by incubation at 37° C. in a humidified
chamber with 5% CO2 for 5 minutes, 30 minutes and 2 hours. At the
end of stimulation, cells were washed once with PBS and lysed with RIPA
buffer (Sigma) containing phosphatase and protease inhibitors according
to the manufacturer's directions. 1 ug to 20 ug of cell lysate was
applied to MULTI-SPOT Phospho (Ser 473)/Total Akt Assay plate (Meso Scale
Discovery, K15100D-1). After a short spin at low speed, plate was
incubated at room temperature for 2 hour with shaking Washes, preparation
of detection antibody and preparation of Read Buffer were performed
according to manufacture's suggestions. Plate was analyzed with SECTOR
Imager 6000 and the percentage of phospho-Akt signal was calculated as
manufacture recommended ((2*phosphor Akt signal)/(phosphor Akt
signal+total Akt signal))*100.

Example 21

MSD Phospho-Akt Detection of Endogenous REG4 Expressing Cells

[0186] 5-10 μg of KM12 and PC3 cell lysate were applied to MULTI-SPOT
Phospho (Ser 473)/Total Akt Assay plate (Meso Scale Discovery, K15100D-1)
following REG4 and negative control siRNA knock down for 24 hours, 48
hours and 72 hours. After a short spin at low speed, plate was incubated
at room temperature for 2 hour with shaking Washes, preparation of
detection antibody and preparation of Read Buffer were performed
according to manufacture's suggestions. Plate was analyzed with SECTOR
Imager 6000 and the percentage of phospho-Akt signal was calculated as
manufacture recommended.

[0187] Treatment with REG4 protein resulted in an increase of
phosphorylated proteins. Conversely, siRNA REG4 knockdown in PC3 and KM12
cells resulted in a decrease of phosphorylation.

Example 22

Endothelial Cell Tubule Formation Assay

[0188] Microtiter plates were coated with 50 μA of ECMatrix provided as
part of the In vitro Angiogenesis assay kit (Chemicon ECM625). Harvested
HUVEC cells (Lonza) as per supplier's instructions in 25% EGM media and
prepared a cell suspension containing 20,000 cell/100 μl media. Added
100 μl cell suspension per well. Added 50 μl 3×
concentrations of proteins in 25% EGM to each well. After 18 hrs cells
were stained with the MTT kit (Roche). 20 ul MTT reagent was added to the
well and plates were incubated for 3 hours at 37° C. Images taken
with a 4× objective using a Spot camera. 4 images were taken for
each well at 4× magnification using Discovery-1. Images for each
condition (12 images) were analyzed using the Angiogenesis module
provided with the Metamorph software version 6.1r6.

[0190] A two-tailed Student's t test was used for statistical analysis of
comparative data using the GraphPad Prism program. Data were expressed as
means of at least three independent experiments ±SE, with P<0.05
considered statistically significant.

Table 10 provides a brief description of the sequences in the sequence
listing.